Methods and agents for screening for compounds capable of modulating VEGF expression

ABSTRACT

The invention relates to the fields of screening assays and compounds and methods for altering protein expression and levels of protein. In particular, the invention includes assays to screen for agents capable of modulating expression of VEGF and agents capable of modulating VEGF expression.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of InternationalApplication PCT/US04/01643, filed Jan. 21, 2004, entitled Methods forIdentifying Compounds that Modulate Untranslated Region-Dependent GeneExpression and Methods of Using Same, under 35 U.S.C. §120. TheInternational Application PCT/US04/01643, claims the benefit of andincorporates by reference U.S. Provisional Application No. 60/441,637,filed on Jan. 21, 2003. The entirety of these applications, includingthe sequence listing, is hereby incorporated by reference.

INCORPORATION OF SEQUENCE LISTING

[0002] A paper copy of the Sequence Listing and a computer readable formof the sequence listing on diskette, containing the file named“19025.014.seqlist.txt”, which is 16,435 bytes in size (measured inMS-DOS), and which was recorded on May 24, 2004, are herein incorporatedby reference.

BACKGROUND OF THE INVENTION

[0003] Aberrant angiogenesis plays a role in the pathogenesis ofnumerous diseases, including malignant, ischemic, inflammatory andimmune disorders (reviewed in Matter, Drug Discovery Today, 6:1005-1024(2001); Yancopoulos et al, Nature, 407:242-248(2000); Carmeliet, Nat.Med., 9(6):653-660 (2003); Ferrara, Semin. Oncol., 29(6Suppl 16):10-14(2002)). Vascular Endothelial Growth Factor (VEGF), an angiogenesisregulator, plays a central role in angiogenesis. In particular, VEGF isan important factor in the pathogenesis of cancers, diabetic retinopathy(DR), and exudative macular degeneration (reviewed in Tandle et al.,Clin. Adv. in Hemat. and Oncol., 1(1):41-48 (2003); Ferrara et al., Nat.Med., 5(12):1359-1364 (1999); Matter, supra; Carmeliet supra; Kerbel etal., Nat. Rev. Cancer, 2(10):727-739 (2002); Witmer et al., Prog. Retin.Eye Res., 22(1):1-29 (2003); Clark et al., Nat. Rev. Drug Discovery,2:448-459 (2003); Ferrara (2002), supra; Thomas, J. Biol. Chem.,271:603-606 (1996); Gerber et al., Development, 126:1149-1159 (1999);which are hereby incorporated by reference), the last two of which areleading causes of blindness in the United States.

[0004] The expression of VEGF is regulated by a number of factors andagents including cytokines, growth factors, steroid hormones andchemicals, and mutations that modulate the activity of oncogenes such asras or the tumor suppressor gene VHL (Maxwell et al., Nature,399:271-275 (1999); Rak et al., Cancer Res., 60:490-498 (2000)). Inpart, VEGF expression is regulated after transcription by sequences inboth the 5′- and 3′-untranslated regions (UTRs) of its mRNA (Ikeda etal., J. Biol. Chem., 270:19761-19766 (1995); Stein et al., Mol. Cell.Biol., 18:3112-3119 (1998); Levy et al. J. Biol. Chem., 271:2746-2753(1996); Huez et al., Mol. Cell. Biol., 18:6178-6190 (1998); Akiri etal., Oncogene, 17:227-236 (1998)). The VEGF 5′ UTR is unusually long andGC rich, and it contains an internal ribosomal entry site (IRES) that isreported to mediate a unique, cap-independent mode of translationinitiation. The VEGF 3′ UTR harbors multiple AU-rich stabilitydeterminants that have been shown to be associated with VEGF mRNAturnover rates.

[0005] Initiation of translation of the VEGF mRNA is reported to beunique under hypoxic conditions in that it is mediated via an internalribosome entry site (IRES) within the VEGF 5′ UTR (Stein et al., supra;Levy et al., supra; Huez et al., supra; Akiri et al., supra). Underhypoxic conditions, cap-dependent translation is dramatically impairedand the translation of the VEGF mRNA occurs through its cap-independentIRES. Initiation of translation of most eukaryotic mRNA iscap-dependent. IRES-mediated translation initiation becomes predominantwhen components of the translation initiation complex becomerate-limiting, e.g., during hypoxia (Mitchell et al., Mol. Cell.,1(3):757-771 (2003)).

[0006] Several investigators have used in vitro and bicistronicstrategies, often in conjunction with deletion mutants, to studyregulation of VEGF. Prats and colleagues reported the occurrence ofcap-independent translation of human VEGF by virtue of an IRES. Fromthese studies, they postulated the presence of two IRESs, a first IRES(IRES A) located within 300 nucleotides of the initiation codon and asecond IRES (IRES B) located in the upstream half of the 5′-UTR. Huez etal., supra. In Stein et al., supra., deletion mutants in dicistronic andmonocistronic constructs were used to identify sequences of the VEGF 5′UTR required for maximal IRES activity. Keshet and colleagues reportedan increase in IRES activity from a 163-nucleotide sequence derived froma VEGF 5′ UTR, which is possibly an artifact from RT-PCR amplification,relative to the entire full-length VEGF 5′ UTR. Stein et al., supra.Goodall and colleagues reported a deletion analysis of IRES residuestoward the 3′ end of the mouse VEGF 5′ UTR, and speculated that, foroptimal IRES activity, the upstream half of the VEGF 5′ UTR isnecessary. Miller et al., supra.

[0007] The present invention provides, for the first time, a negativeregulator of post-transcriptional regulation (NeRP) located in the 5′UTR of VEGF. Removal of a NeRP from the VEGF 5′ UTR results in increasedtranslation of an operably linked gene dependent on the presence of aPTCRE of the present invention.

SUMMARY OF THE INVENTION

[0008] The present invention includes a nucleic acid constructcomprising a nucleic acid sequence encoding a reporter polypeptide,where the nucleic acid sequence encoding a reporter polypeptide isoperably linked to a NeRP, the NeRP is operably linked to a PTCRE, thePTCRE is not SEQ ID NO: 3, and expression of the reporter polypeptide iscapable of being modulated relative to in an absence of the NeRP.

[0009] The present invention also includes a nucleic acid moleculecomprising a nucleic acid sequence encoding a reporter polypeptide and aVEGF 5′ UTR nucleic acid sequence in an absence of SEQ ID NO: 4.

[0010] The present invention also includes a nucleic acid moleculecomprising a nucleic acid sequence encoding a reporter polypeptideoperably linked to a VEGF 5′ UTR in an absence of SEQ ID NO: 4.

[0011] The present invention also includes a nucleic acid moleculecomprising a nucleic acid sequence encoding a reporter polypeptide,where the nucleic acid sequence encoding a reporter polypeptide isoperably linked downstream of a UTR containing a NeRP, and the UTR isnot operably upstream of SEQ ID NO: 3.

[0012] The present invention also includes a heterogeneous population ofnucleic acid molecules, where the heterogeneous population comprises areporter nucleic acid sequence, and the nucleic acid sequence encoding areporter polypeptide is operably linked to a VEGF 5′ UTR in an absenceof NeRP1 (SEQ ID NO: 4).

[0013] The present invention also includes a substantially purifiednucleic acid molecule comprising between 95% and 99% sequence identitywith a nucleic acid molecule of SEQ ID NO: 3, a fragment thereof, or acomplement of either.

[0014] The present invention also includes a substantially purifiednucleic acid molecule consisting of SEQ ID NO: 3, a fragment thereof, ora complement of either.

[0015] The present invention also includes a substantially purifiednucleic acid molecule consisting of a first nucleic acid sequence linkedto a heterologous nucleic acid sequence encoding a polypeptide, wherethe first nucleic acid sequence is selected from the group consisting ofSEQ ID NO: 3, a fragment thereof, and a complement of either.

[0016] The present invention also includes a substantially purifiednucleic acid molecule comprising between 95% and 99% sequence identitywith a nucleic acid molecule of SEQ ID NO: 4, a fragment thereof, or acomplement of either.

[0017] The present invention also includes a substantially purifiednucleic acid molecule of a nucleic acid sequence selected from a groupconsisting of SEQ ID NO: 4, a fragment thereof, and a complement ofeither.

[0018] The present invention also includes a substantially purifiednucleic acid molecule consisting of a first nucleic acid sequence linkedto a heterologous nucleic acid sequence encoding a polypeptide, wherethe first nucleic acid sequence is selected from the group consisting ofSEQ ID NO: 4, a fragment thereof, and a complement of either.

[0019] The present invention also includes a method of making a nucleicacid construct to screen for a compound comprising: a) providing a mainORF downstream of a promoter in the nucleic acid construct; b) operablylinking a VEGF 5′ UTR in an absence of SEQ ID NO: 4 upstream of the mainORF; and c) operably linking a VEGF 3′ UTR downstream of the main ORF.

[0020] The present invention also includes a method of screening in vivofor a compound that modulates UTR-dependent expression comprising: a)providing a cell having a nucleic acid molecule comprising a promoterupstream from a VEGF 5′ UTR in an absence of SEQ ID NO: 4, where theVEGF 5′ UTR in an absence of SEQ ID NO: 4 is upstream from a nucleicacid sequence encoding a reporter polypeptide, and the nucleic acidsequence encoding a reporter polypeptide is upstream from a VEGF 3′ UTR;b) contacting the cell with a compound; c) producing a nucleic acidmolecule that contains a nucleic acid sequence encoding a reporterpolypeptide and does not contain SEQ ID NO: 4; and d) detecting thereporter polypeptide.

[0021] The present invention also includes a method of screening invitro for a compound that modulates UTR-affected expression comprising:a) providing an in vitro translation system; b) contacting the in vitrotranslation system with a compound and a nucleic acid moleculecomprising a VEGF 5′ UTR in an absence of SEQ ID NO: 4, where the VEGF5′ UTR in an absence of SEQ ID NO: 4 is upstream from a nucleic acidsequence encoding a reporter polypeptide and the nucleic acid sequenceencoding a reporter polypeptide is upstream from a VEGF 3′ UTR; andc)detecting the reporter polypeptide in vitro.

[0022] The present invention also includes a method of expressing anucleic acid molecule in a cell comprising: a) providing a nucleic acidmolecule to a cell, where the nucleic acid molecule comprises a nucleicacid sequence encoding a reporter polypeptide flanked by VEGF UTRs in anabsence of SEQ ID NO: 4; and b) detecting the reporter polypeptide.

[0023] The present invention also includes a method of screening for acompound that modulates protein expression through a mainORF-independent, UTR-affected mechanism comprising: a) growing a stablecell line having a reporter gene operably linked to a VEGF 5′ UTR in anabsence of SEQ ID NO: 4; b) comparing the stable cell line in a presenceof a compound relative to the stable cell line in an absence of thecompound; and c) selecting for the compound that modulates proteinexpression through a main ORF-independent, UTR-affected mechanism.

[0024] The present invention also includes a method of screening for acompound that modulates protein expression through a mainORF-independent, UTR-affected mechanism comprising: a) growing a stablecell line having a main ORF operably linked to a VEGF 5′ UTR in anabsence of SEQ ID NO: 4; b) comparing the stable cell line in thepresence of a compound relative to the stable cell line in the absenceof the compound; and c) selecting for the compound that modulatesprotein expression through a main ORF-independent, UTR-affectedmechanism.

[0025] The present invention also includes a method of screening for acompound that modulates protein expression through a VEGF-independent,UTR-affected mechanism comprising: a) substituting in vivo a VEGF genewith a reporter gene, where a UTR consisting of SEQ ID NO: 3 is operablylinked to the reporter gene, and the substitution occurs in adifferentiated cell; b) growing the differentiated cell; and c)selecting for the compound that modulates protein expression of thereporter gene through a main ORF-independent, UTR-affected mechanism.

[0026] The present invention also includes a method of screening for acompound that modulates protein expression through a mainORF-independent, UTR-affected mechanism comprising: a) substituting invivo a main ORF with a reporter gene, where a 5′ UTR is operably linkedto the reporter gene and consists of SEQ ID NO: 3, and the substitutionoccurs in a differentiated cell; b) growing the differentiated cell; andc) selecting for the compound that modulates protein expression of thereporter gene through a main ORF-independent, UTR-affected mechanism.

[0027] The present invention also includes a method of screening for acompound that modulates protein expression through a UTR-affectedmechanism comprising: a) providing a stable cell line having a reportergene operably linked to a VEGF 5′ UTR in an absence of SEQ ID NO: 4,where the stable cell line mimics post-transcriptional regulation of aVEGF gene found in vivo in presence of the compound; b) maintaining thestable cell line; and c) selecting for the compound that modulatesprotein expression of the reporter gene through a UTR-affectedmechanism.

[0028] The present invention also includes a method of screening for acompound that modulates protein expression through a UTR-affectedmechanism comprising: a) providing a stable cell line having a main ORFencoding a reporter polypeptide operably linked to a VEGF 5′ UTR in anabsence of SEQ ID NO: 4, where the stable cell line mimicspost-transcriptional regulation of a VEGF gene found in vivo in apresence of a compound; b) maintaining the stable cell line; and c)selecting for the compound that modulates protein expression of the mainORF through a UTR-affected mechanism.

[0029] The present invention also includes a method of screening for acompound that modulates protein expression through a UTR-affectedmechanism mediating the effect of a NeRP comprising: a) growing a stablecell line having a reporter gene operably linked to a 5′ VEGF UTR in anabsence of a NeRP1 (SEQ ID NO: 4); b) comparing the stable cell line ina presence of a compound relative to in an absence of the compound,where the compound does not modulate UTR-dependent expression if the 5′VEGF UTR in an absence of a NeRP1 (SEQ ID NO: 4) is operably linked to areporter gene; and c) selecting for the compound that modulates proteinexpression of the reporter gene through a UTR-affected mechanismmediating the effect of a NeRP.

[0030] The present invention also includes a method of screening for acompound that modulates protein expression through a UTR-affectedmechanism mediating the effect of a NeRP comprising: a) growing a stablecell line having a main ORF encoding a reporter polypeptide operablylinked to a 5′ VEGF UTR in an absence of a NeRP1 (SEQ ID NO: 4); b)comparing the stable cell line in the presence of a compound relative tothat in the absence of the compound, where the compound does notmodulate UTR-dependent expression if the 5′ VEGF UTR in an absence of aNeRP1 (SEQ ID NO: 4) is operably linked to a main ORF; and c) selectingfor the compound that modulates protein expression of the main ORFthrough a UTR-affected mechanism mediating the effect of a NeRP.

[0031] The present invention also includes a method of screening for acompound that modulates protein expression through a UTR-affectedmechanism mediating the effect of a NeRP comprising: a) growing a stablecell line having a reporter gene operably linked to a UTR having a NeRP1(SEQ ID NO: 4); b) comparing the stable cell line in a presence of acompound relative to in an absence of the compound, where the compoundmodulates UTR-dependent expression if a NeRP1 (SEQ ID NO: 4) is operablylinked to a reporter gene; and c) selecting for. the compound thatmodulates protein expression of the reporter gene through a UTR-affectedmechanism mediating the effect of a NeRP.

[0032] The present invention also includes a method of screening for acompound that modulates protein expression through a UTR-affectedmechanism mediating the effect of a NeRP comprising: a) growing a stablecell line having a main ORF encoding a reporter polypeptide operablylinked to a UTR having a NeRP1 (SEQ ID NO: 4); b) comparing the stablecell line in a presence of a compound relative to in an absence of thecompound, where the compound modulates UTR-dependent expression if aNeRP1 (SEQ ID NO: 4) is operably linked to a main ORF encoding areporter polypeptide; and c) selecting for the compound that modulatesprotein expression of the main ORF encoding a reporter polypeptidethrough a UTR-affected mechanism mediating the effect of the NeRP.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033]FIG. 1 sets forth a sequence alignment of VEGF 5′ UTR for mouse,rat, and human.

[0034]FIG. 2 sets forth an example of a compound that inhibits VEGFexpression over a concentration range.

[0035]FIG. 3 sets forth an example of UTR-dependent inhibition of VEGFexpression, where VEGF expression is dependent on one or more VEGF UTRs.

[0036]FIG. 4A sets forth an example of effective inhibition of VEGFproduction in tumor tissue.

[0037]FIG. 4B sets forth the inhibition of human VEGF production in vivoby a compound.

[0038]FIG. 5 sets forth a schematic of a 5′ VEGF UTR and restrictionsites therein. VEGF 5′ UTR is amplified from human genomic DNA by twoseparate PCR reactions. In the overlap region of 5′ UTR1 and 5′ UTR2, aunique enzyme site BamHI is used to assemble the full-length 5′ UTR insubsequent cloning.

[0039]FIG. 6 sets forth a schematic representation of dicistronicplasmids that can be used for transfection experiments.P2luc/5UTR-ΔNeRP1 is a dicistronic plasmid containing VEGF 5′ UTR1, inwhich nucleotides 337 to 1083 of the VEGF cDNA are fused to the fireflyluciferase coding sequence; P2luc/5UTR-FL is generated by subcloningVEGF 5′ UTR2 into the plasmid p2luc/vegf5utr1 between SalI and BamHI;plasmid p2luc/5UTR-Δ51-476 is derived from p2luc/5UTR-FL by removing theNheI fragment (nt 51 to 746); plasmid p2luc/5UTR-Δ476-1038 is derivedfrom p2luc/vegf5utr-fl by removing the sequence from BamHI site to the3′end of 5′ UTR; plasmid p2luc/5UTR-Δ1-476 is derived from p2luc/5UTR-FLby removing the sequence from BamHI to the 5′ end of 5′ UTR. P2luc-e canbe used as a negative control in this study.

[0040]FIG. 7 sets forth results identifying the presence of a VEGF IRESdomain (a PTCRE) and a NeRP in the VEGF mRNA 5′ UTR. Reporter geneexpression is analyzed by monitoring luciferase activity.

DESCRIPTION OF THE NUCLEIC ACID SEQUENCES

[0041] SEQ ID NO: 1 sets forth a full-length VEGF 5′ UTR.

[0042] SEQ ID NO: 2 sets forth an open reading frame encoding VEGF.

[0043] SEQ ID NO: 3 sets forth a PTCRE1, a 702 nucleotide region of aVEGF 5′ UTR.

[0044] SEQ ID NO: 4 sets forth a NeRP1, a 336 nucleotide region of aVEGF 5′ UTR.

[0045] SEQ ID NO: 5 sets forth a PTCRE2, a 485 nucleotide region of aVEGF 5′ UTR.

[0046] SEQ ID NO: 6 sets forth a PTCRE3, a 556 nucleotide region of aVEGF 5′ UTR.

[0047] SEQ ID NO: 7 sets forth a PTCRE4, a 294 nucleotide region of aVEGF 5′ UTR.

[0048] SEQ ID NO: 8 sets forth a PTCRE5, a 194 nucleotide region of aVEGF 5′ UTR.

[0049] SEQ ID NO: 9 sets forth a NeRP2, a 476 nucleotide region of aVEGF 5′ UTR.

[0050] SEQ ID NO: 10 sets forth a NeRP3, a 554 nucleotide region of aVEGF 5′ UTR.

[0051] SEQ ID NO: 11 sets forth a NeRP4, a 51 nucleotide region of aVEGF 5′ UTR.

[0052] SEQ ID NO: 12 sets forth a NeRP5, a 91 nucleotide region of aVEGF 5′ UTR.

[0053] SEQ ID NO: 13 sets forth a NeRP6, a 335 nucleotide region of aVEGF 5′ UTR.

[0054] SEQ ID NO: 14 sets forth a NeRP7, a 332 nucleotide region of aVEGF 5′ UTR.

[0055] SEQ ID NO: 15 sets forth a NeRP8, a 331 nucleotide region of aVEGF 5′ UTR.

[0056] SEQ ID NO: 16 sets forth a NeRP9, a 330 nucleotide region of aVEGF 5′ UTR.

[0057] SEQ ID NO: 17 sets forth a NeRP10, a 329 nucleotide region of aVEGF 5′ UTR.

[0058] SEQ ID NO: 18 sets forth a NeRP11, a 328 nucleotide region of aVEGF 5′ UTR.

[0059] SEQ ID NO: 19 sets forth a NeRP12, a 327 nucleotide region of aVEGF 5′ UTR.

[0060] SEQ ID NO: 20 sets forth a NeRP13, a 326 nucleotide region of aVEGF 5′ UTR.

[0061] SEQ ID NO: 21 sets forth a NeRP14, a 316 nucleotide region of aVEGF 5′ UTR.

[0062] SEQ ID NO: 22 sets forth a NeRP15, a 306 nucleotide region of aVEGF 5′ UTR.

[0063] SEQ ID NO: 23 sets forth a NeRP16, a 296 nucleotide region of aVEGF 5′ UTR.

[0064] SEQ ID NO: 24 sets forth a NeRP17, a 286 nucleotide region of aVEGF 5′ UTR.

[0065] SEQ ID NO: 25 sets forth a NeRP18, a 276 nucleotide region of aVEGF 5′ UTR.

[0066] SEQ ID NO: 26 sets forth a NeRP19, a 266 nucleotide region of aVEGF 5′ UTR.

Definitions

[0067] As used herein, the term “construct” refers to an artificiallymanipulated nucleic acid molecule.

[0068] As used herein, the term “heterologous” refers to ingredients orconstituents of dissimilar or diverse origin.

[0069] As used herein, the term “mammalian cancer cell” or “mammaliantumor cell” refers to a cell derived from a mammal that proliferatesinappropriately.

[0070] As used herein, the term “main ORF-independent mechanism” refersto a cellular pathway or process, wherein at least one step relates togene expression and is not dependent on the nucleic acid sequence of themain open reading frame.

[0071] As used herein, the term “reporter gene” refers to any gene whoseexpression can be measured.

[0072] As used herein, the term “RNA induced gene silencing, or RNAinterference (RNAi)” refers to the mechanism of double-stranded RNA(dsRNA) introduced into a system to reduce protein expression ofspecific genetic sequence.

[0073] As used herein, the term “specifically bind” means that acompound binds to another compound in a manner different from a similartype of compounds, e.g. in terms of affinity, avidity, and the like. Ina non-limiting example, more binding occurs in the presence of acompeting reagent, such as casein. In another non-limiting example,antibodies that specifically bind a target protein should provide adetection signal at least 2-, 5-, 10-, or 20-fold higher relative to adetection signal provided with other molecules when used in Westernblots or other immunochemical assays. In an alternative non-limitingexample, a nucleic acid can specifically bind its complementary nucleicacid molecule. In another non-limiting example, a transcription factorcan specifically bind a particular nucleic acid sequence.

[0074] As used herein, the term “secondary structure” means thealpha-helical, beta-sheet, random coil, beta turn structures and helicalnucleic acid structures that occur in proteins, polypeptides, nucleicacids, compounds comprising modified nucleic acids, compounds comprisingmodified amino acids, and other types of compounds as a result of, atleast, the compound's composition.

[0075] As used herein, the term “non-peptide therapeutic agent” andanalogous terms include, but are not limited to organic or inorganiccompounds (i.e., including heteroorganic and organometallic compoundsbut excluding proteins, polypeptides and nucleic acids).

[0076] As used herein, the term “UTR” refers to the untranslated regionof a mRNA.

[0077] As used herein, the term “untranslated region-dependentexpression” or “UTR-dependent expression” refers to the regulation ofgene expression through UTRs at the level of mRNA expression, i.e.,after transcription of the gene has begun until the protein or the RNAproduct(s) encoded by the gene has degraded or excreted.

[0078] As used herein, the term “vector” refers to a nucleic acidmolecule used to introduce a nucleic acid sequence in a cell ororganism. The entirety of the International Application PCT/US04/01643,filed Jan. 21, 2004, including the sequence listing, is herebyincorporated by reference.

DETAILED DESCRIPTION OF THE INVENTION

[0079] The present invention includes and utilizes the fact that anuntranslated region (UTR) is capable of modulating expression of a geneand that such modulation of expression is capable of being altered ormodulated by the addition of compounds. In a preferred embodiment, a UTRis a region of a RNA that is not translated into protein. In a morepreferred embodiment, a UTR is a flanking region of the RNA transcriptthat is not translated into the targeted protein, and can include a 5′UTR that has a short, putative open reading frame. In a most preferredembodiment, the UTR is a 5′ UTR, i.e., upstream of the coding region, ora 3′ UTR, i.e., downstream of the coding region.

[0080] Moreover, the present invention includes and provides agents andmethods useful in screening for a compound capable of modulating geneexpression and also hybrid molecules.

[0081] Nucleic Acid Agents and Constructs

[0082] One skilled in the art may refer to general reference texts fordetailed descriptions of known techniques discussed herein or equivalenttechniques. These texts include Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Inc. (1995); Sambrook et al.,Molecular Cloning, A Laboratory Manual (2d ed.), Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1989); Birren et al., Genome Analysis:A Laboratory Manual, volumes 1 through 4, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (1997-1999). These texts can, of course, also bereferred to in making or using an aspect of the invention.

[0083] UTRs

[0084] The present invention includes nucleic acid molecules with UTRsthat comprise or consist of a post-transcriptional regulatory element(PTCRE) including SEQ ID NO: 3, a negative regulator of a PTCRE (NeRP)including SEQ ID NO: 4, and fragments and complements of all.

[0085] A PTCRE of the present invention can differ from any of theresidues in SEQ ID NO: 3 in that the nucleic acid sequence has beendeleted, substituted, or added in a manner that does not alter thefunction. In another aspect of the present invention, a PTCRE of thepresent invention consists or comprises SEQ ID NO: 5, and fragments andcomplements of all. In another aspect of the present invention, a PTCREof the present invention consists or comprises SEQ ID NOs: 6-8, andfragments and complements of all.

[0086] A PTCRE of the present invention can differ from any of theresidues in an untranslated region selected from the group consisting ofa nucleic acid sequence consisting or comprising SEQ ID NO: 3 and SEQ IDNOs: 5-8 in that the nucleic acid sequence has been deleted,substituted, or added in a manner that does not alter the function.

[0087] A NeRP of the present invention can differ from any of theresidues in SEQ ID NO: 4 in that the nucleic acid sequence has beendeleted, substituted, or added in a manner that does not alter thefunction.

[0088] In another aspect of the present invention, a NeRP of the presentinvention consists or comprises SEQ ID NO: 9, and fragments andcomplements of all. In another aspect of the present invention, a NeRPof the present invention consists or comprises SEQ ID NOs: 10-12, andfragments and complements of all.

[0089] A NeRP of the present invention can differ from any of theresidues in a UTR selected from the group consisting of a nucleic acidsequence consisting or comprising SEQ ID NO: 4 and SEQ ID NOs: 9-12 inthat the nucleic acid sequence has been deleted, substituted, or addedin a manner that does not alter the function. In one aspect, a NeRP isnot a full-length sequence of a target UTR. In a preferred aspect, aNeRP is not a full-length VEGF 5′ UTR.

[0090] In one embodiment, a NeRP can be a nucleic acid sequence with asingle base deletion at any location of SEQ ID NO: 4. Therefore, a NeRPof the invention may be 335 bases. In another embodiment, a NeRPincludes a nucleic acid sequence with two or more bases deleted from anylocation of SEQ ID NO: 4. In another embodiment, a NeRP includes 3, 4,5, 6, 7, 8, 9 or 10 residue deletions at any location of SEQ ID NO: 4.In another embodiment, a NeRP includes the remaining nucleic acidsequence resulting from the deletion of 20, 30, 40, 50, 60, or 70residues from any location of SEQ ID NO: 4. In light of thespecification, a NeRP of the present invention may be produced bycontiguous, noncontiguous, or a combination of contiguous deletions andnoncontiguous deletions of SEQ ID NO: 4 in a manner that does not alterthe function of the NeRP.

[0091] In a preferred embodiment, when a nucleic acid molecule, whichincludes a NeRP and a PTCRE, has been deleted, substituted, or added toin a manner that removes the negative regulation of a PTCRE, thesecondary structure of the remaining nucleic acid molecule is altered ina manner comparable to the alteration in the secondary structure of afull-length VEGF 5′ UTR when SEQ ID NO: 4 is deleted. In a morepreferred embodiment, a NeRP of the present invention has a secondarystructure comparable to the secondary structure of SEQ ID NO: 4.

[0092] In an embodiment of the present invention, the presence of a NeRPcan be detected by the deletion, insertion or alteration of one or morepseudoknots from a larger nucleic acid molecule. In another embodiment,the deletion, insertion or alteration of a stem-loop structure from alarger nucleic acid molecule of the present invention results in a NeRPof the present invention. Programs such as mfold, (see the world wideweb at bioweb.pasteur.fr/seqanal/interfaces/mfold-simple.html) genebee(see the world wide web at genebee.msu.su/) may be used to ascertain thesecondary structure of SEQ ID NO: 4 and other nucleic acid molecules ofthe present invention. Other programs or methods well known to those ofskill in the art can also be employed.

[0093] The present invention also includes a NeRP that has a tertiarystructure comparable to the tertiary structure formed by SEQ ID NO: 4.Tertiary structure can be determined by, for example, crystallographyand phylogenetic covariation (as reviewed in Martin I, et al., BiochimBiophys Acta. 2003 Jul. 11;1614(1):97-103; Heinemann U., et al., BiolChem. 1996 July-August;377(7-8):447-54).

[0094] The present invention provides nucleic acid molecules thathybridize to the above-described nucleic acid molecules. In a preferredaspect, the nucleic acid molecule hybridizes to a nucleic acid moleculeselected from the group consisting of a nucleic acid sequence consistingor comprising SEQ ID NOs: 3-12, and complements thereof. Nucleic acidhybridization is a technique well known to those of skill in the art ofDNA manipulation. The hybridization properties of a nucleic acidmolecule are an indication of their similarity or identity. The nucleicacid molecules preferably hybridize, under moderate or high stringencyconditions, with a nucleic acid sequence selected from SEQ ID NO: 5 andcomplements thereof. Fragments of these sequences are also contemplated.

[0095] In another aspect, the nucleic acid molecules preferablyhybridize, under moderate or high stringency conditions, with a nucleicacid sequence selected from the group consisting of SEQ ID NO: 5 and itscomplement. The hybridization conditions typically involve nucleic acidhybridization in about 0.1× to about 10×SSC (diluted from a 20×SSC stocksolution containing 3 M sodium chloride and 0.3M sodium citrate, pH 7.0in distilled water), about 2.5× to about 5×Denhardt's solution (dilutedfrom a 50×stock solution containing 1% (w/v) bovine serum albumin, 1%(w/v) Ficoll® (Amersham Biosciences Inc., Piscataway, N.J.), and 1%(w/v) polyvinylpyrrolidone in distilled water), about 10 mg/ml to about100 mg/ml salmon sperm DNA, and about 0.02% (w/v) to about 0.1% (w/v)SDS, with an incubation at about 20° C. to about 70° C. for severalhours to overnight.

[0096] In a preferred aspect, the moderate stringency hybridizationconditions are provided by 6×SSC, 5×Denhardt's solution, 100 mg/mlsalmon sperm DNA, and 0.1% (w/v) SDS, with an incubation at 55° C. forseveral hours. The moderate stringency wash conditions are about 0.02%(w/v) SDS, with an incubation at about 55° C. overnight. In a morepreferred aspect, the high stringency hybridization conditions are about2×SSC, about 3×Denhardt's solution, and about 10 mg/ml salmon sperm DNA.The high stringency wash conditions are about 0.05% (w/v) SDS, with anincubation at about 65° C. overnight.

[0097] In an embodiment, the nucleic acid molecule comprises a nucleicacid sequence that is greater than 85% identical, and more preferablygreater than 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%identical to a nucleic acid sequence selected from the group consistingof SEQ ID NOs: 3-12, complements thereof, and fragments of any of thesesequences.

[0098] The percent identity is preferably determined using the “BestFit” or “Gap” program of the Sequence Analysis Software Package™(Version 10; Genetics Computer Group, Inc., University of WisconsinBiotechnology Center, Madison, Wis.). “Gap” utilizes the algorithm ofNeedleman and Wunsch to find the alignment of two sequences thatmaximizes the number of matches and minimizes the number of gaps.“BestFit” performs an optimal alignment of the best segment ofsimilarity between two sequences and inserts gaps to maximize the numberof matches using the local homology algorithm of Smith and Waterman. Thepercent identity calculations may also be performed using the Megalignprogram of the LASERGENE bioinformatics computing suite (defaultparameters, DNASTAR Inc., Madison, Wis.). The percent identity is mostpreferably determined using the “Best Fit” program using defaultparameters.

[0099] Fragment nucleic acid molecules can contain significant portionsof, or indeed most of, SEQ ID NO: 3. In an embodiment, the fragments arebetween about 160 and 250 consecutive residues, about 260 and about 350consecutive residues, about 360 and about 400 consecutive residues, orabout 460 and 500 consecutive residues long of a nucleic molecule of thepresent invention. In another embodiment, the fragment comprises atleast 170, 300, 500, or 600 consecutive residues of SEQ ID NO: 3. In aparticularly preferred embodiment, a fragment nucleic acid molecule iscapable of selectively hybridizing to SEQ ID NO: 3.

[0100] In one embodiment, a PTCRE comprises or consists of SEQ ID NO: 3.In another embodiment, a PTCRE comprises or consists of a fragment ofSEQ ID NO: 3. In a preferred embodiment, a PTCRE can share identity withbetween particular mammals, including, but not limited to human, mouse,and rat. In another preferred embodiment, a PTCRE is unique to a humanVEGF 5′ UTR PTCRE, a non-limiting example of which is SEQ ID NO: 3.

[0101] Fragment nucleic acid molecules can contain significant portionsof SEQ ID NO: 4. In another embodiment of the present invention, nucleicacid molecules can comprise or consist of significant portions of SEQ IDNO: 4. In an embodiment, the fragments are between about 40 and about 90consecutive residues, about 100 and about 150 consecutive residues,about 160 and about 250 consecutive residues, or about 260 and 325consecutive residues long of a nucleic molecule of the presentinvention. In another embodiment, the fragment comprises at least 90,150, 250, or 325 consecutive residues of SEQ ID NO: 4. In a preferredembodiment, a fragment nucleic acid molecule is capable of selectivelyhybridizing to SEQ ID NO: 4.

[0102] In one embodiment, a NeRP comprises or consists of SEQ ID NO: 4.In another embodiment, a NeRP comprises or consists of a fragment of SEQID NO: 4. In a preferred embodiment, a NeRP can share identity withbetween particular mammals, including, but not limited to human, mouse,and rat. In another preferred embodiment, a NeRP is unique to a humanVEGF 5′ UTR NeRP, a non-limiting example of which is SEQ ID NO: 4.

[0103] Any of a variety of methods may be used to obtain one or more ofthe above-described nucleic acid molecules of the present invention.Automated nucleic acid synthesizers may be employed for this purpose. Inlieu of such synthesis, the disclosed nucleic acid molecules may be usedto define a pair of primers that can be used with the polymerase chainreaction (PCR) to amplify and obtain any desired nucleic acid moleculeor fragment.

[0104] Short nucleic acid sequences having the ability to specificallyhybridize to complementary nucleic acid sequences may be produced andutilized in the present invention, e.g., as probes to identify thepresence of a complementary nucleic acid sequence in a given sample.Alternatively, the short nucleic acid sequences may be used asoligonucleotide primers to amplify or mutate a complementary nucleicacid sequence using PCR technology. These primers may also facilitatethe amplification of related complementary nucleic acid sequences (e.g.,related sequences from other species).

[0105] Use of these probes or primers may greatly facilitate theidentification of transgenic cells or organisms that contain thepresently disclosed structural nucleic acid sequences. Such probes orprimers may also, for example, be used to screen cDNA, mRNA, or genomiclibraries for additional nucleic acid sequences related to or sharinghomology with the presently disclosed promoters and structural nucleicacid sequences. The probes may also be PCR probes, which are nucleicacid molecules capable of initiating a polymerase activity while in adouble-stranded structure with another nucleic acid.

[0106] A primer or probe is generally complementary to a portion of anucleic acid sequence that is to be identified, amplified, or mutatedand of sufficient length to form a stable and sequence-specific duplexmolecule with its complement. The primer or probe preferably is about 10to about 200 residues long, more preferably is about 10 to about 100residues long, even more preferably is about 10 to about 50 residueslong, and most preferably is about 14 to about 30 residues long.

[0107] The primer or probe may, for example without limitation, beprepared by direct chemical synthesis, by PCR (U.S. Pat. Nos. 4,683,195and 4,683,202), or by excising the nucleic acid specific fragment from alarger nucleic acid molecule. Various methods for determining thesequence of PCR probes and PCR techniques exist in the art.Computer-generated searches using programs such as Primer3(www-genome.wi.mit. edu/cgi-bin/primer/primer3.cgi), STSPipeline(www-genome.wi.mit.edu/cgi-bin/www-STS_Pipeline), or GeneUp (Pesole etal., BioTechniques 25:112-123, 1998), for example, can be used toidentify potential PCR primers.

[0108] Furthermore, sequence comparisons can be done to find nucleicacid molecules of the present invention based on secondary structurehomology. Several methods and programs are available to predict andcompare secondary structures of nucleic acid molecules, for example,GeneBee (available on the world wide web atgenebee.msu.su/services/rna2_reduced.html); the Vienna RNA Package(available on the world wide web at tbi.univie.ac.at/˜ivo/RNA/);SstructView (available on the world wide web at the Stanford MedicalInformatics website, under: projects/helix/sstructview/home.html anddescribed in “RNA Secondary Structure as a Reusable Interface toBiological Information Resources.” 1997. Gene vol. 190GC59-70). Forexample, comparisons of secondary structure are preformed in Le et al.,A common RNA structural motif involved in the internal initiation oftranslation of cellular mRNAs. 1997. Nuc. Acid. Res. vol. 25(2):362-369.

[0109] Constructs of the Present Invention

[0110] The present invention includes and provides nucleic acidconstructs. It is understood that any of the constructs and othernucleic acid agents of the present invention can be either DNA or RNA.In a preferred embodiment, a construct can be a nucleic acid moleculehaving a UTR, a coding sequence, or both. In another embodiment, aconstruct is composed of at least one UTR of the present invention, asequence encoding a reporter polypeptide, and a vector. Moreover, any ofthe nucleic acid molecules of the present invention can be used incombination with a method of the present invention.

[0111] Vectors

[0112] Exogenous genetic material may be introduced into a host cell byuse of a vector or construct designed for such purpose. Any of thenucleic acid sequences of the present invention can be incorporated intoa vector or construct of the present invention. A vector or construct ofthe present invention includes, without limitation, linear or closedcircular plasmids. A vector system may be a single vector or plasmid ortwo or more vectors or plasmids that together contain the total DNA tobe introduced into the genome of the host. In a preferred embodiment, avector contains a promoter functional in mammalian cells or bacteria orboth. Means for preparing vectors or constructs are well known in theart.

[0113] Vectors suitable for replication in mammalian cells may includeviral replicons, or sequences that insure integration of the appropriatesequences encoding HCV epitopes into the host genome. For example,another vector used to express foreign DNA is vaccinia virus. Suchheterologous DNA is generally inserted into a gene that is non-essentialto the virus, for example, the thymidine kinase gene (tk), which alsoprovides a selectable marker. Expression of the HCV polypeptide thenoccurs in cells or animals that are infected with the live recombinantvaccinia virus.

[0114] In general, plasmid vectors containing replicon and controlsequences that are derived from species compatible with the host cellare used in connection with bacterial hosts. The vector ordinarilycarries a replication site, as well as marking sequences that arecapable of providing phenotypic selection in transformed cells. Forexample, E. coli is typically transformed using a construct with abackbone derived from a vector, such as pBR322, which contains genes forampicillin and tetracycline resistance and thus provides easy means foridentifying transformed cells. The pBR322 plasmid, or other microbialplasmid or phage, also generally contains, or is modified to contain,promoters that can be used by the microbial organism for expression ofthe selectable marker genes.

[0115] In a preferred embodiment of the present invention, an expressionvector can be a high-level mammalian expression vector designed torandomly integrate into the genome, for example, pCMR1. In anotherpreferred embodiment of the present invention, an expression vector canbe a high-level mammalian expression vector designed tosite-specifically integrate into the genome of cells. For example, pMCP1can site-specifically integrate into the genome of cells geneticallyengineered to contain the FRT site-specific recombination site via theFlp recombinase (see, e.g., Craig, 1988, Ann. Rev. Genet. 22: 77-105;and Sauer, 1994, Curr. Opin. Biotechnol. 5: 521-527).

[0116] Promoters

[0117] A construct can include a promoter, e.g., a recombinant vectortypically comprises, in a 5′ to 3′ orientation: a promoter to direct thetranscription of a nucleic acid molecule of interest.

[0118] In a preferred aspect of the present invention, a construct caninclude a mammalian promoter and can be used to express a nucleic acidmolecule of choice. As used herein, a “mammalian promoter” refers to apromoter functional in a mammalian cell derived from a mammalian cell orboth. A number of promoters that are active in mammalian cells have beendescribed in the literature. A promoter can be selected on the basis ofthe cell type into which the vector will be inserted.

[0119] A preferred promoter of the present invention is a VEGF promoter.In addition to VEGF promoters described previously, other promotersequences can be utilized in a construct or other nucleic acid molecule.Suitable promoters include, but are not limited to, those describedherein.

[0120] Suitable promoters for mammalian cells are known in the art andinclude viral promoters, such as those from Simian Virus 40 (SV40), Roussarcoma virus (RSV), adenovirus (ADV), cytomegalovirus (CMV), and bovinepapilloma virus (BPV), and the parvovirus B19p6 promoter as well asmammalian cell-derived promoters. A number of viral-based expressionsystems can be used to express a reporter gene in mammalian host cells.For example, if an adenovirus is used as an expression vector, sequencesencoding a reporter gene can be ligated into an adenovirustranscription/translation complex comprising the late promoter andtripartite leader sequence.

[0121] Other examples of preferred promoters include tissue-specificpromoters and inducible promoters. Other preferred promoters include thehematopoietic stem cell-specific, e.g., CD34, glucose-6-phosphotase,interleukin-1 alpha, CD11c integrin gene, GM-CSF, interleukin-5R alpha,interleukin-2, c-fos, h-ras and DMD gene promoters. Other promotersinclude the herpes thymidine kinase promoter, and the regulatorysequences of the metallothionein gene.

[0122] Inducible promoters suitable for use with bacteria hosts includethe β-lactamase and lactose promoter systems, the arabinose promotersystem, alkaline phosphatase, a tryptophan (trp) promoter system andhybrid promoters such as the tac promoter. However, other knownbacterial inducible promoters are suitable. Promoters for use inbacterial systems also generally contain a Shine-Dalgarno sequenceoperably linked to the DNA encoding the polypeptide of interest.

[0123] A promoter can also be selected on the basis of their regulatoryfeatures, e.g., enhancement of transcriptional activity, inducibility,tissue specificity, and developmental stage-specificity. A promoter canwork in vitro, for example the T7-promoter. Particularly preferredpromoters can also be used to express a nucleic acid molecule of thepresent invention in a nonhuman mammal. Additional promoters that may beutilized are described, for example, in Bernoist and Chambon, Nature290:304-310 (1981); Yamamoto et al., Cell 22:787-797 (1980); Wagner etal., PNAS 78:1441-1445 (1981); Brinster et al., Nature 296:39-42 (1982).

[0124] Reporter Genes

[0125] As used herein, a “reporter gene” is any gene whose expressioncan be measured. In a preferred embodiment, a reporter gene does nothave any UTRs. In a more preferred embodiment, a reporter gene is acontiguous open reading frame. In another preferred embodiment, areporter gene can have a previously determined reference range ofdetectable expression.

[0126] Constructs of the invention can comprise one or more reportergenes fused to one or more UTRs. For example, specific RNA sequences,RNA structural motifs, and/or RNA structural elements that are known orsuspected to modulate UTR-dependent expression of a target gene can befused to the reporter gene. A reporter gene of the present inventionencoding a protein, a fragment thereof, or a polypeptide, can also belinked to a propeptide encoding region. A propeptide is an amino acidsequence found at the amino terminus of a proprotein or proenzyme.Cleavage of the propeptide from the proprotein yields a maturebiochemically active protein. The resulting polypeptide is known as apropolypeptide or proenzyme (a zymogen in some cases). Propolypeptidesare generally inactive and can be converted to mature activepolypeptides by catalytic or autocatalytic cleavage of the propeptidefrom the propolypeptide or proenzyme.

[0127] A reporter gene can express a selectable or screenable marker.Selectable markers may also be used to select for organisms or cellsthat contain exogenous genetic material. Examples of such include, butare not limited to: a neo gene, which codes for kanamycin resistance andcan be selected for using kanamycin, GUS, neomycin phosphotransferase II(nptII), or an antibiotic resistance coding sequence. Screenable markerscan be used to monitor expression. Exemplary screenable markers include:green fluorescent protein (GFP), luciferase (LUX), a β-glucuronidase oruidA gene (GUS) which encodes an enzyme for which various chromogenicsubstrates are known; a β-lactamase gene, a gene which encodes an enzymefor which various chromogenic substrates are known (e.g., PADAC, achromogenic cephalosporin); a luciferase gene; a tyrosinase gene, whichencodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinonewhich in turn condenses to melanin; and α-galactosidase, which can turna chromogenic α-galactose substrate.

[0128] Included within the terms “selectable or screenable marker genes”are also genes that encode a secretable marker whose secretion can bedetected as a means of identifying or selecting for transformed cells.Examples include markers that encode a secretable antigen that can beidentified by antibody interaction, or even secretable enzymes, whichcan be detected utilizing their inherent properties. Secretable proteinsfall into a number of classes, including small, diffusible proteinswhich are detectable, (e.g., by ELISA), or small active enzymes whichare detectable in extracellular solution (e.g., α-amylase, β-lactamase,phosphinothricin transferase). Other possible selectable or screenablemarker genes, or both, are apparent to those of skill in the art.

[0129] A reporter gene can express a fusion protein. As such, the fusionprotein can be a fusion of any reporter gene operably linked to anothergene, or fragment thereof. For instance, the expressed fusion proteincan provide a “tagged” epitope to facilitate detection of the fusionprotein, such as GST, GFP, FLAG, or polyHIS. Such fusions preferablyencode between 1 and 50 amino acids, more preferably between 5 and 30additional amino acids, and even more preferably between 5 and 20 aminoacids. In one embodiment, a fusion protein can be a fusion protein thatincludes in whole or in part of a VEGF protein sequence.

[0130] Alternatively, the fusion can provide regulatory, enzymatic, cellsignaling, or intercellular transport functions. For example, a sequenceencoding a signal peptide can be added to direct a fusion protein to aparticular organelle within a eukaryotic cell. Such fusion partnerspreferably encode between 1 and 1000 additional amino acids, morepreferably between 5 and 500 additional amino acids, and even morepreferably between 10 and 250 amino acids.

[0131] The present invention also provides for a reporter gene flankedby one or more untranslated regions (e.g., the 5′ UTR, 3′ UTR, or boththe 5′ UTR and 3′ UTR of the target gene). In addition, the presentinvention provides for a reporter gene flanked by one or more UTRs of atarget gene, where the UTR contains one or more mutations (e.g., one ormore substitutions, deletions and/or additions). In a preferredembodiment, the reporter gene is flanked by both 5′ and 3′ UTRs so thatcompounds that interfere with an interaction between the 5′ and 3′ UTRscan be identified.

[0132] In another preferred embodiment, a stable hairpin secondarystructure is inserted into the UTR, preferably the 5′ UTR of the targetgene. For example, in cases where the 5′ UTR possesses IRES activity,the addition of a stable hairpin secondary structure in the 5′ UTR canbe used to separate cap-dependent from cap-independent translation (see,e.g., Muhlrad et al., 1995, Mol. Cell. Biol. 15(4):2145-56, thedisclosure of which is incorporated by reference in its entirety). Inanother embodiment, an intron is inserted into a UTR (preferably, the 5′UTR) or at the 5′ end of an ORF of a target gene. For example, but notby limitation, in cases where an RNA possesses instability elements, anintron, e.g., the human elongation factor one alpha (EF-1 alpha) firstintron, can be cloned into a UTR (preferably, the 5′ UTR) or a 5′ end ofthe ORF to increase expression (see, e.g., Kim et al., 2002, JBiotechnol 93(2):183-7, the disclosure of which is incorporated byreference in its entirety). In a preferred embodiment, both a stablehairpin secondary structure and an intron are added to the reporter geneconstruct. In a more preferred embodiment, the stable hairpin secondarystructure is cloned into the 5′ UTR and the intron is added at the 5′end of the main ORF of the reporter gene.

[0133] The reporter gene can be positioned such that the translation ofthat reporter gene is dependent upon the mode of translation initiation,such as, but not limited to, cap-dependent translation orcap-independent translation (i.e., translation via an internal ribosomeentry site). Alternatively, where the UTR contains an upstream openreading frame, the reporter gene can be positioned such that thereporter protein is translated only in the presence of a compound thatshifts the reading frame of the UTR so that the formerly untranslatedopen reading frame is then translated.

[0134] The reporter gene constructs can be monocistronic ormulticistronic. A multicistronic reporter gene construct may encode 2,3, 4, 5, 6, 7, 8, 9, 10 or more, or in the range of 2-5, 5-10 or 10-20reporter genes. For example, a dicistronic reporter gene constructcomprising, in the following order going downstream, a promoter, a firstreporter gene, a 5′ UTR of a target gene, a second reporter gene andoptionally, a 3′ UTR of a target gene. In such a reporter construct, thetranscription of both reporter genes is capable of being driven by thepromoter. In this example construct, the present invention includes thetranslation of the mRNA from the first reporter gene by a cap-dependentscanning mechanism and the translation of the mRNA from the secondreporter gene by a cap-independent mechanism, for example by an IRES. Insuch a case, the IRES-dependent translation of a mRNA of the secondreporter gene can be normalized against the cap-dependent translation.

[0135] Reporter genes can be expressed in vitro or in vivo. In vivoexpression can be in a suitable bacterial or eukaryotic host. Suitablemethods for expression are described by Sambrook et al., MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989); Haymes et al.,Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington,D.C. (1985); or similar texts. Fusion protein or peptide molecules ofthe invention are preferably produced via recombinant means. Theseproteins and peptide molecules can be derivatized to containcarbohydrate or other moieties (such as keyhole limpet hemocyanin,etc.).

[0136] Linked

[0137] As used herein, linked means physically linked, operably linked,flanked, or any of these in combination.

[0138] As used herein, physically linked means that the physicallylinked nucleic acid sequences are located on the same nucleic acidmolecule, for example a promoter can be physically linked to a reportergene as part of a construct. A physical linkage can be proximal, andeither direct or indirect. In a preferred embodiment, the promoter isoperably linked and physically linked to a nucleic acid sequence of thepresent invention.

[0139] A preferred embodiment of the present invention also provides forspecific nucleic acid molecules containing a reporter gene flanked byone or more UTRs of a target gene. In this preferred embodiment, the oneor more UTRs of a target gene can be physically linked, operably linked,or operably and physically linked to the reporter gene. The presentinvention also provides for a reporter gene flanked by one or more UTRsof a target gene, where one or more of the UTRs contains one or moremutations (e.g., one or more of each substitution, deletion, addition,or any combination of each). In a more-preferred embodiment of thepresent invention, a reporter gene is flanked by a 5′ UTR of a VEGF genecontaining one or more deletions. In a most preferred embodiment, areporter gene is flanked by and operably linked to a 5′ VEGF UTR in theabsence of a NeRP.

[0140] In a preferred embodiment, the reporter gene is flanked by both5′ and 3′ UTRs of one or more target genes so that compounds thatinterfere with an interaction between the 5′ and 3′ UTRs can beidentified. In a more preferred embodiment, the reporter gene is flankedby a 5′ and 3′ UTRs of one target gene, and the reporter gene isphysically, operably, or physically and operably linked to the UTRs ofone target gene. In a most preferred embodiment, a reporter gene isproximally linked, either directly or indirectly, to one or more UTRs ofa target gene. If the reporter gene is directly linked to a UTR of atarget gene, the last nucleic acid residue of the reporter gene ischemically bonded to the first nucleic acid residue of the UTR of atarget gene. If the reporter gene is proximally linked indirectly to aUTR of a target gene, the last nucleic acid residue of the reporter geneis not chemically bonded to the first nucleic acid residue of the UTR ofa target gene and the last nucleic acid residue of the reporter gene canbe about 3 or greater than 5 but less than 20. If the reporter gene isdirectly linked to a UTR of a target gene at any time during reportergene processing, such as after a splicing event, the reporter gene isdirectly linked to the UTR.

[0141] UTRs

[0142] Agents and constructs of the invention include nucleic acidmolecules with an untranslated region (UTR). In a preferred aspect, aUTR refers to a UTR of an mRNA, i.e. the region of the mRNA that is nottranslated into protein. In a preferred embodiment, a UTR contains oneor more regulatory elements that modulate UTR-dependent regulation ofgene expression. In a particularly preferred embodiment, a UTR is a 5′UTR, i.e., upstream of the coding region, or a 3′ UTR, i.e., downstreamof the coding region. In another particularly preferred embodiment, the5′ UTR includes a VEGF promoter. In a more preferred embodiment, the 5′UTR includes a VEGF promoter and a PTCRE.

[0143] As used herein, a “main ORF” is a nucleic acid sequence,including sequence contained in deoxyribonucleic acid and ribonucleicacid molecules, having an open reading frame that can be translated.Examples of a main ORF include a reporter gene, a target gene, and acontrol gene. As used herein, a “target gene” can be any gene. In apreferred embodiment, a target gene is a gene operatively linkeddownstream of a VEGF 5′ UTR containing an upstream open reading frame(“uORF”). In another embodiment, a target gene can be a VEGF main ORF.In a preferred embodiment, a target gene is a gene containing a uORF. Ina particularly preferred embodiment, a target gene is a gene havinggreater than 50% identity greater than 200 residues with respect to aVEGF gene. As used herein, a “control gene” can be any gene. In apreferred embodiment, a control gene is a gene operatively linkeddownstream of a VEGF 5′ UTR that does not contain a NeRP.

[0144] A UTR of the present invention can be operatively, physically, oroperatively and physically linked to a reporter gene. In a preferredembodiment of the present invention, a UTR of the present invention isphysically linked to a reporter gene. The physical, operable, orphysical and operable linkage may be upstream, downstream, or internalto the reporter gene. As used herein, operably linked means that theoperably linked nucleic acid sequences exhibit their deserved function.For example, a promoter can be operably linked to a reporter gene.

[0145] In a preferred aspect of the present invention, a UTR of thepresent invention is a VEGF 5′ UTR physically linked upstream of areporter gene. In a particularly preferred embodiment, VEGF 5′ UTRcontains or consists of SEQ ID NO: 3 and is physically linked downstreamof a reporter gene. In another embodiment, a VEGF 5′ UTR does notcontain or consist of a NeRP and is physically and operatively linkedupstream of a reporter gene. In a more particularly preferredembodiment, VEGF 5′ UTR does not contain or consist of a NeRP and doescontain or consist of a PTCRE and is physically and operatively linkedupstream of a reporter gene.

[0146] In a preferred embodiment of the present invention, a UTR of thepresent invention is physically linked upstream to a reporter gene andanother UTR is physically linked downstream of the reporter gene. In aparticularly preferred embodiment, a UTR of the present inventioncontains or consists of SEQ ID NO: 3 and is physically and operativelylinked upstream of a reporter gene and a VEGF 3′ UTR is physically andoperatively linked downstream of a reporter gene.

[0147] In a preferred embodiment of the present invention, a UTR of thepresent invention is physically linked to reporter gene containing anintron. In a more preferred embodiment of the present invention, a UTRof the present invention containing SEQ ID NO: 3 is physically linked toa reporter gene containing an intron. In a preferred embodiment of thepresent invention, a UTR of the present invention is physically linkedupstream of a reporter gene and contains an intron internal to the UTR.

[0148] In a preferred embodiment of the present invention, a UTR of thepresent invention is physically linked upstream of a reporter gene and aUTR is physically linked downstream of the reporter gene. In a morepreferred embodiment of the present invention, a VEGF 5′ UTR of thepresent invention containing a SEQ ID NO: 3 is physically linkedupstream of a reporter gene and a VEGF 3′ UTR is physically linkeddownstream of the reporter gene.

[0149] PTCREs and NeRPs

[0150] As referred to herein, a PTCRE is a post-transcriptionalregulatory element that modulates expression of a target gene. In oneaspect, a PTCRE is not a full-length sequence of a target UTR. In apreferred aspect, a PTCRE is not a full-length VEGF 5′ UTR. In oneembodiment, a PTCRE in one target gene can have primary nucleic acidsequence similarity to a PTCRE in a different target gene.Alternatively, there may not be any primary nucleic acid sequencesimilarity in PTCREs of similar function. In a preferred embodiment, aPTCRE in one target gene can have a secondary, tertiary, or secondaryand tertiary structure similar to a PTCRE in a different target gene.Examples of PTCREs include, but are not limited to, IRES elements,upstream ORFs, and AREs.

[0151] In one embodiment, a PTCRE is a nucleic acid sequence in a UTR,which modulates UTR-dependent expression. A PTCRE can be a nucleic acidsequence selected from the group consisting of an iron response element(“IRE”), Internal ribosome entry site (“IRES”), upstream open readingframe (“uORF”), male specific lethal element (“MSL-2”), G quartetelement, and 5′-terminal oligopyrimidine tract (“TOP”), AU-rich element(“ARE”), selenocysteine insertion sequence (“SECIS”), histone stem loop,cytoplasmic polyadenylation element (“CPE”), nanos translational controlelement, amyloid precursor protein element (“APP”), translationalregulation element (“TGE”)/direct repeat element (“DRE”), Bruno element(“BRE”), or a 15-lipoxygenase differentiation control element(“15-LOX-DICE”). In an alternative embodiment, a PTCRE is not an IRESfrom VEGF. In another embodiment, PTCRE is not be an IRES. In apreferred embodiment, a PTCRE is SEQ ID NO: 3. In a most preferredembodiment, a PTCRE is not a NeRP and the PTCRE does not contain orconsist of a NeRP.

[0152] A NeRP is a nucleic acid sequence in a UTR, which modulatesPTCRE-dependent expression in a NeRP-dependent mechanism. In oneembodiment of the present invention, a NeRP regulates an IRES. In apreferred embodiment, a NeRP suppresses IRES-dependent expression of agene. In a most preferred embodiment, a NeRP is SEQ ID NO: 4.Alternatively, a NeRP can modulate PTCRE-dependent expression, where thePTCRE suppresses gene expression, so that the NeRP is capable ofincreasing gene expression by a NeRP-dependent mechanism. In analternative embodiment of the invention, a NeRP mimics an IRES.

[0153] A NeRP of the present invention can differ from any of theresidues in SEQ ID NO: 4 in that the nucleic acid sequence has beendeleted, substituted, or added in a manner that does not alter thefunction. In another aspect of the present invention, a NeRP of thepresent invention consists or comprises SEQ ID NO: 9, and fragments andcomplements of all. In another aspect of the present invention, a NeRPof the present invention consists or comprises SEQ ID NOs: 10-12, andfragments and complements of all.

[0154] While the present invention is directed, in part, to VEGF 5′UTRs, PTCREs of the present invention can be located in any positionwithin a construct and not limited to the 5′ UTR region of a construct.A PTCRE of the present invention can be operatively, physically, oroperatively and physically linked to a UTR. In an alternative embodimentof the present invention, a PTCRE of the present invention is a UTR ofthe present invention.

[0155] While the present invention is directed, in part, to a NeRP inthe VEGF 5′ UTR, NeRPs of the present invention can be located in anyposition within a construct and not limited to the VEGF 5′ UTR region ofa construct. A NeRP of the present invention can be operatively,physically, or operatively and physically linked to a UTR. In analternative embodiment of the present invention, a NeRP of the presentinvention is upstream of a PTCRE of the present invention.

[0156] In a preferred embodiment, a PTCRE of the present invention islocated between about 1 to about 100 residues upstream from theinitiation codon of an open reading frame in a mRNA, between about 150to about 250 residues upstream from the initiation codon, or betweenabout 300 to about 500 residues upstream from the initiation codon. In amost preferred embodiment, the untranslated region is about 1 residueupstream from the initiation codon.

[0157] In a preferred embodiment, a NeRP of the present invention isbetween about 1000 to about 500 residues upstream from a PTCRE, betweenabout 500 to about 100 residues upstream from a PTCRE, or between about100 to about 60 residues upstream from a PTCRE. In another embodiment, aPTCRE is within about 1000 residues upstream from the 5′ end of a mainORF, about 500 residues upstream from the 5′ end of a main ORF, orwithin about 200 residues upstream from the 5′ end of a main ORF, orabout 100 residues upstream from the 5′ end of a main ORF. In anotherembodiment, a PTCRE is within the main ORF and between about 1000 toabout 500 residues upstream from the 3′ end of a main ORF, between about500 to about 100 residues upstream from the 3′ end of a main ORF, orbetween about 100 to about 60 residues upstream from the 3′ end of amain ORF. In a most preferred embodiment, the PTCRE is within 30residues upstream from the 5′ end of a main ORF.

[0158] Constructs of the present invention can have more or fewercomponents than described above. For example, constructs of the presentinvention can include genetic elements, including but not limited to, 3′transcriptional terminators, 3′ polyadenylation signals, otheruntranslated nucleic acid sequences, transit or targeting sequences,selectable or screenable markers, promoters, enhancers, and operators,as desired. Constructs of the present invention can also contain apromoterless gene that may utilize an endogenous promoter upon insertioninto a host cell chromosome.

[0159] Alternatively, sequences encoding nucleic acid molecules of thepresent invention can be cloned into a vector for the production of anmRNA probe. Such vectors are known in the art, are commerciallyavailable, and can be used to synthesize RNA probes in vitro by additionof labeled nucleotides and an appropriate RNA polymerase such as T7, T3,or SP6. These procedures can be conducted using a variety ofcommercially available kits (for example, Amersham Biosciences Inc.,Piscataway, N.J.; and Promega Co, Madison, Wis.).

[0160] Modulation of Gene Expression by Nucleic Acid Molecules of thePresent Invention

[0161] Modulation of gene expression can result in more or less geneexpression. Many approaches for modulating gene expression using nucleicacid molecules of the present invention are known to one skilled in theart. For example, over-expression of a gene product can be the resultfrom transfection of a construct of the present invention into amammalian cell. Similarly, down-regulation can be the result fromtransfection of a construct of the present invention into a mammaliancell. Other non-limiting examples include anti-sense techniques like RNAinterference (RNAi), transgenic animals, hybrids, and ribozymes. Thefollowing examples are provided by way of illustration, and are notintended to be limiting of the present invention.

[0162] Cellular Mechanisms

[0163] As used herein, the term “UTR-dependent expression” refers to theregulation of gene expression through UTRs at the level of mRNAexpression, i.e., after transcription of the gene has begun until theprotein or the RNA product(s) encoded by the gene has degraded. In apreferred embodiment, the term “UTR-dependent expression” refers to theregulation of mRNA stability or translation. In a more preferredembodiment, the term “UTR-dependent expression” refers to the regulationof gene expression through regulatory elements present in an UTR(s).Altering the sequence of a PTCRE within a UTR of target gene may changethe amount of UTR-dependent expression observed for that target gene.

[0164] As used herein, a “UTR-affected mechanism” is a cellularmechanism that discriminates between UTRs based on their nucleic acidsequence or based on properties that are a function of their sequencesuch as the secondary, tertiary, or quaternary structure or otherassociated factors. Modulation of the UTR-dependent expression of atarget gene can be due to a change in how a UTR-affected mechanism actson the target gene. For example, a UTR in a target gene can contain anIRES, which affects target gene expression via a UTR-affected mechanism.

[0165] In a preferred embodiment, a UTR-affected mechanism can be a mainORF-independent mechanism. As used herein, a “main ORF-independentmechanism” refers to a cellular pathway or process, wherein at least onestep relates to gene expression and is not dependent on the nucleic acidsequence of the main open reading frame. In a preferred embodiment, aUTR-affected mechanism is a main ORF-independent, UTR-affectedmechanism.

[0166] In order to exclude the possibility that a particular compound isfunctioning solely by modulating the expression of a target gene in anUTR-independent manner, one or more mutations may be introduced into theUTRs operably linked to a reporter gene and the effect on the expressionof the reporter gene in a reporter gene-based assay described herein canbe determined. For example, a reporter gene construct comprising the 5′UTR of a target gene may be mutated by deleting a fragment of the 5′ UTRof the target gene or substituting a fragment of the 5′ UTR of thetarget gene with a fragment of the 5′ UTR of another gene and measuringthe expression of the reporter gene in the presence and absence of acompound that has been identified in a screening assays of the presentinvention or of an assay well known to the skilled artisan. If thedeletion of a fragment of the 5′ UTR of the target gene or thesubstitution of a fragment of the 5′ UTR of the target gene with afragment of the 5′ UTR of another gene affects the ability of thecompound to modulate the expression of the reporter gene, then thefragment of the 5′ UTR deleted or substituted plays a role in theregulation of the reporter gene expression and the regulation, at leastin part, in an UTR-dependent manner.

[0167] Alternatively or in conjunction with tests described above, thepossibility that a particular compound is functioning solely bymodulating the expression of a target gene in an UTR-independent mannercan be determined by changing the vector utilized as a reporterconstruct. The UTRs flanked by a reporter gene from the first reporterconstruct in which an effect on reporter gene expression was detectedfollowing exposure to a compound may be inserted into a new reporterconstruct that has, e.g., different transcriptional regulation elements(e.g., a different promoter) and a different selectable marker. Thelevel of reporter gene expression in the presence of the compound can becompared to the level of reporter gene expression in the absence of thecompound or in the presence of a control (e.g., PBS). If there is nochange in the level of expression of the reporter gene in the presenceof the compound relative to the absence of the compound or in thepresence of a control, then the compound probably is functioning in anUTR-independent manner.

[0168] Compounds, identified in assays of the present invention, thatare capable of modulating UTR-dependent expression of a target gene (forconvenience referred to herein as a “lead” compound) can be furthertested for UTR-dependent binding to the target RNA (which contains atleast one UTR, and preferably at least one element of an UTR, forexample a PTCRE). Furthermore, by assessing the effect of a compound ontarget gene expression, cis-acting elements, i.e., specific nucleotidesequences, that are involved in UTR-dependent expression may beidentified. RNA binding assays, subtraction assays, and expressedprotein concentration and activity assays are examples of methods todetermine UTR-dependent expression of a gene.

[0169] Hybrids

[0170] In one aspect of the present invention, a hybrid of a compoundand a PTCRE or a NeRP of the present invention is a hybrid formedbetween two non-identical molecules. In a preferred aspect, a hybrid canbe formed between two nucleic acid molecules. For example, a hybrid canbe formed between two ribonucleic acid molecules, between a ribonucleicacid molecule and a deoxyribonucleic acid molecule, or betweenderivatives of either. In alternative embodiment, a hybrid can be formedbetween a nucleic acid of the present invention and a non-nucleic acidmolecule. In a preferred embodiment, a hybrid can be formed between anucleic acid molecule and a non-nucleic acid molecule, for example, apolypeptide or a non-peptide therapeutic agent.

[0171] Ribozymes

[0172] In one aspect of the present invention, the activity orexpression of a gene is regulated by designing trans-cleaving catalyticRNAs (ribozymes) specifically directed to a nucleic acid molecule of thepresent invention, for example, SEQ ID NO: 3 and SEQ ID NOs: 5-8. In analternate aspect, the activity or expression of a gene is regulated bydesigning trans-cleaving catalytic RNAs (ribozymes) specificallydirected to a nucleic acid molecule of the present invention, forexample, SEQ ID NO: 4 and SEQ ID NOs: 9-12.

[0173] Ribozymes are RNA molecules possessing endoribonuclease activity.Ribozymes are specifically designed for a particular target, and thetarget message contains a specific nucleotide sequence. They areengineered to cleave any RNA species site-specifically in the backgroundof cellular RNA. The cleavage event renders the mRNA unstable andprevents protein expression. Importantly, ribozymes can be used toinhibit expression of a gene of unknown function for the purpose ofdetermining its function in an in vitro or in vivo context, by detectinga phenotypic effect.

[0174] One commonly used ribozyme motif is the hammerhead, for which thesubstrate sequence requirements are minimal. Design of the hammerheadribozyme, and the therapeutic uses of ribozymes, are disclosed in Usmanet al., Current Opin. Strict. Biol. 6:527-533 (1996). Ribozymes can alsobe prepared and used as described in Long et al., FASEB J. 7:25 (1993);Symons, Ann. Rev. Biochem. 61:641 (1992); Perrotta et al., Biochem.31:16-17 (1992); Ojwang et al., PNAS 89:10802-10806 (1992); and U.S.Pat. No. 5,254,678.

[0175] Ribozyme cleavage of HIV-I RNA, methods of cleaving RNA usingribozymes, methods for increasing the specificity of ribozymes, and thepreparation and use of ribozyme fragments in a hammerhead structure aredescribed in U.S. Pat. Nos. 5,144,019; 5,116,742; and 5,225,337 andKoizumi et al., Nucleic Acid Res. 17:7059-7071 (1989). Preparation anduse of ribozyme fragments in a hairpin structure are described byChowrira and Burke, Nucleic Acids Res. 20:2835 (1992). Ribozymes canalso be made by rolling transcription as described in Daubendiek andKool, Nat. Biotechnol. 15(3):273-277 (1997).

[0176] The hybridizing region of the ribozyme may be modified or may beprepared as a branched structure as described in Horn and Urdea, NucleicAcids Res. 17:6959-67 (1989). The basic structure of the ribozymes mayalso be chemically altered in ways familiar to those skilled in the art,and chemically synthesized ribozymes can be administered as syntheticoligonucleotide derivatives modified by monomeric units. In atherapeutic context, liposome mediated delivery of ribozymes improvescellular uptake, as described in Birikh et al., Eur. J. Biochem.245:1-16 (1997).

[0177] Ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”) such as the onewhich occurs naturally in Tetrahymena thermophila (known as the IVS, orL-19 IVS RNA) and which has been extensively described by Thomas Cechand collaborators (Zaug et al., Science 224:574-578 (1984); Zaug andCech, Science 231:470-475 (1986); Zaug et al., Nature, 324:429-433(1986); WO 88/04300; Been and Cech, Cell 47:207-216 (1986)). TheCech-type ribozymes have an eight base pair active site which hybridizesto a target RNA sequence whereafter cleavage of the target RNA takesplace. The invention encompasses those Cech-type ribozymes which targeteight base-pair active site sequences that are present in a target gene.

[0178] Ribozymes can be composed of modified oligonucleotides (e.g., forimproved stability, targeting, etc.) and should be delivered to cellswhich express the target gene in vivo. A preferred method of deliveryinvolves using a DNA construct “encoding” the ribozyme under the controlof a strong constitutive pol III or pol II promoter, so that transfectedcells will produce sufficient quantities of the ribozyme to destroyendogenous messages and inhibit translation. Because ribozymes, unlikeantisense molecules, are catalytic, a lower intracellular concentrationis required for efficiency.

[0179] Using the nucleic acid sequences of the invention and methodsknown in the art, ribozymes are designed to specifically bind and cutthe corresponding mRNA species. Ribozymes thus provide a means toinhibit the expression of any of the proteins encoded by the disclosednucleic acids or their full-length genes. The full-length gene need notbe known in order to design and use specific inhibitory ribozymes. Inthe case of a nucleic acid or cDNA of unknown function, ribozymescorresponding to that nucleotide sequence can be tested in vitro forefficacy in cleaving the target transcript. Those ribozymes that effectcleavage in vitro are further tested in vivo. The ribozyme can also beused to generate an animal model for a disease, as described in Birikhet al., Eur. J. Biochem. 245:1-16 (1997). An effective ribozyme is usedto determine the function of the gene of interest by blocking itstranscription and detecting a change in the cell. Where the gene isfound to be a mediator in a disease, an effective ribozyme is designedand delivered in a gene therapy for blocking transcription andexpression of the gene.

[0180] Therapeutic and functional genomic applications of ribozymesbegin with knowledge of a portion of the coding sequence of the gene tobe inhibited. Thus, for many genes, a partial nucleic acid sequenceprovides adequate sequence for constructing an effective ribozyme. Atarget cleavage site is selected in the target sequence, and a ribozymeis constructed based on the 5′ and 3′ nucleotide sequences that flankthe cleavage site. Retroviral vectors are engineered to expressmonomeric and multimeric hammerhead ribozymes targeting the mRNA of thetarget coding sequence. These monomeric and multimeric ribozymes aretested in vitro for an ability to cleave the target mRNA. A cell line isstably transduced with the retroviral vectors expressing the ribozymes,and the transduction is confirmed by Northern blot analysis andreverse-transcription polymerase chain reaction (RT-PCR). The cells arescreened for inactivation of the target mRNA by such indicators asreduction of expression of disease markers or reduction of the geneproduct of the target mRNA.

[0181] Cells and Organisms

[0182] Nucleic acid molecules that may be used in cell transformation ortransfection can be any of the nucleic acid molecules of the presentinvention. Nucleic acid molecules of the present invention can beintroduced into a cell or organism. In a preferred aspect, the cell isselected from the group consisting of cells that do not express VEGF,cells that express VEGF, or cells that express VEGF conditionally. In amore preferred aspect, the cell is a cancer cell, more preferably acancer cell where VEGF is overexpressed relative to a non-transformedcell.

[0183] A host cell strain can be chosen for its ability to modulate theexpression of the inserted sequences, to process an expressed reportergene in the desired fashion, or based on the expression levels of anendogenous or heterologous VEGF gene. Mammalian cell lines available ashosts for expression are known in the art and include many immortalizedcell lines available from the American Type Culture Collection (ATCC,Manassas, Va.), such as HeLa cells, Chinese hamster ovary (CHO) cells,baby hamster kidney (BHK) cells and a number of other cell lines.Non-limiting examples of suitable mammalian host cell lines includethose shown below in Table 1. TABLE 1 Mammalian Host Cell Lines HostCell Origin Source HepG-2 Human Liver Hepatoblastoma ATCC HB 8065 CV-1African Green Monkey Kidney ATCC CCL 70 LLC-MK₂ Rhesus Monkey KidneyATCC CCL 7 3T3 Mouse Embryo Fibroblasts ATCC CCL 92 AV12-664 SyrianHamster ATCC CRL 9595 HeLa Human Cervix Epitheloid ATCC CCL 2 RPMI8226Human Myeloma ATCC CCL 155 H4IIEC3 Rat Hepatoma ATCC CCL 1600 C127IMouse Fibroblast ATCC CCL 1616 293 Human Embryonal Kidney ATCC CRL 1573HS-Sultan Human Plasma Cell Plasmocytoma ATCC CCL 1484 BHK-21 BabyHamster Kidney ATCC CCL 10 CHO-K1 Chinese Hamster Ovary ATCC CCL 61

[0184] In a preferred aspect, cells of the present invention can becells of an organism. In a more preferred aspect, the organism is amammal. In a most preferred aspect, the mammal is a human. In anothermore preferred aspect, the organism is a non-human mammal, preferably amouse, rat, or a chimpanzee. In one aspect of the present invention,cells can be pluripotent or differentiated.

[0185] A nucleic acid of the present invention can be naturallyoccurring in the cell or can be introduced using techniques such asthose described in the art. There are many methods for introducingtransforming DNA segments into cells, but not all are suitable fordelivering DNA to eukaryotic cells. Suitable methods include any methodby which DNA can be introduced into a cell, such as by direct deliveryof DNA, by desiccation/inhibition-mediated DNA uptake, byelectroporation, by agitation with silicon carbide fibers, byacceleration of DNA coated particles, by chemical transfection, bylipofection or liposome-mediated transfection, by calciumchloride-mediated DNA uptake, etc. For example, without limitation,Lipofectamine® (Invitrogen Co., Carlsbad, Calif.) and Fugene®(Hoffmann-La Roche Inc., Nutley, N.J.) can be used for transfection ofnucleic acid molecules, such as constructs and siRNA, into severalmammalian cells. Alternatively, in certain embodiments, accelerationmethods are preferred and include, for example, microprojectilebombardment and the like. Within the scope of this invention, thetransfected nucleic acids of the present invention may be expressedtransciently or stably. Such transfected cells can be in a two- orthree-dimensional cell culture system or in an organism.

[0186] For example, without limitation, the construct may be anautonomously replicating construct, i.e., a construct that exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g., a plasmid, an extrachromosomal element, aminichromosome, or an artificial chromosome. The construct may containany means for assuring self-replication. For autonomous replication, theconstruct may further comprise an origin of replication enabling theconstruct to replicate autonomously in the host cell in question.Alternatively, the construct may be one which, when introduced into thecell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. This integration may bethe result of homologous or non-homologous recombination.

[0187] Integration of a construct or nucleic acid into the genome byhomologous recombination, regardless of the host being considered,relies on the nucleic acid sequence of the construct. Typically, theconstruct contains nucleic acid sequences for directing integration byhomologous recombination into the genome of the host. These nucleic acidsequences enable the construct to be integrated into the host cellgenome at a precise location or locations in one or more chromosomes. Toincrease the likelihood of integration at a precise location, thereshould be preferably two nucleic acid sequences that individuallycontain a sufficient number of nucleic acids, preferably 400 residues to1500 residues, more preferably 800 residues to 1000 residues, which arehighly homologous with the corresponding host cell target sequence. Thisenhances the probability of homologous recombination. These nucleic acidsequences may be any sequence that is homologous with a host cell targetsequence and, furthermore, may or may not encode proteins.

[0188] Stable expression is preferred for long-term, high-yieldproduction of recombinant proteins. For example, cell lines that stablyexpress a reporter gene can be transformed using expression constructsthat can contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate construct. Following the introduction of the construct, cellscan be allowed to grow for 1-2 days in an enriched medium before theyare switched to a selective medium. The purpose of the selectable markeris to confer resistance to selection, and its presence allows growth andrecovery of cells that successfully express the introduced construct.Resistant clones of stably transformed cells can be proliferated usingtissue culture techniques appropriate to the cell type. See, forexample, Animal Cell Culture, R. I. Freshney, ed., 1986.

[0189] Any number of selection systems can be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler et al., 1977. Cell vol.11:223-32) and adenine phosphoribosyltransferase (Lowy et al., 1980 Cellvol. 22:817-23.) genes which can be employed in tk⁻ or apr⁻ (cells,respectively. Also, antimetabolite, antibiotic, or herbicide resistancecan be used as the basis for selection. For example, dhfr confersresistance to methotrexate (Wigler et al., 1980. Proc. Natl. Acad. Sci.vol. 77:3567-70), npt confers resistance to the aminoglycosides,neomycin and G-418 (Colbere-Garapin et al., 1981. J. Mol. Biol. vol.150:1-14), and als and pat confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively. Additional selectablegenes have been described. For example, trpB allows cells to utilizeindole in place of tryptophan, and hisD allows cells to utilize histinolin place of histidine (Hartman & Mulligan, 1988. Proc. Natl. Acad. Sci.vol. 85:8047-51). Visible markers such as anthocyanins, β-glucuronidaseand its substrate GUS, and luciferase and its substrate luciferin, canbe used to identify transformants and to quantify the amount oftransient or stable protein expression attributable to a specificconstruct system (Rhodes et al., 1995. Methods Mol. Biol. vol.55:121-131).

[0190] Although the presence of marker gene expression suggests that areporter gene is also present, its presence and expression may need tobe confirmed. For example, if a sequence encoding a reporter gene isinserted within a marker gene sequence, transformed cells containingsequences that encode a reporter gene can be identified by the absenceof marker gene function. Alternatively, a marker gene can be placed intandem with a sequence encoding a reporter gene under the control of asingle promoter. Expression of the marker gene in response to inductionor selection usually indicates expression of a reporter gene.

[0191] Alternatively, host cells which contain a reporter gene and whichexpress a reporter gene e can be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassayor immunoassay techniques that include membrane, solution, or chip-basedtechnologies for the detection and/or quantification of nucleic acid orprotein. For example, the presence of a reporter gene can be detected byDNA-DNA or DNA-RNA hybridization or amplification using probes orfragments or fragments of polynucleotides encoding a reporter gene.Nucleic acid amplification-based assays involve the use ofoligonucleotides selected from sequences encoding a reporter gene todetect transformants that contain a reporter gene.

[0192] Screening Methods of the Present Invention

[0193] Compound

[0194] The present invention includes methods for screening compoundscapable of modulating gene expression. Any compound can be screened inan assay of the present invention.

[0195] In an embodiment, a compound includes a nucleic acid or anon-nucleic acid, such as a polypeptide or a non-peptide therapeuticagent. In a preferred embodiment, a nucleic acid can be apolynucleotide, a polynucleotide analog, a nucleotide, or a nucleotideanalog. In a more preferred embodiment, a compound can be an antisenseoligonucleotide, which are nucleotide sequences complementary to aspecific DNA or RNA sequence of the present invention. Preferably, anantisense oligonucleotide is at least 11 nucleotides in length, but canbe at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotideslong. Longer sequences also can be used. Antisense oligonucleotides canbe deoxyribonucleotides, ribonucleotides, or a combination of both.

[0196] Nucleic acid molecules, including antisense oligonucleotidemolecules, can be provided in a DNA construct and introduced into acell. Nucleic acid molecules can be anti-sense or sense and double- orsingle-stranded. In a preferred embodiment, nucleic acid molecules canbe interfering RNA (RNAi) or microRNA (miRNA). In a preferredembodiment, the dsRNA is 20-25 residues in length, termed smallinterfering RNAs (siRNA).

[0197] Oligonucleotides can be synthesized manually or by an automatedsynthesizer, by covalently linking the 5′ end of one nucleotide with the3′ end of another nucleotide with non-phosphodiester internucleotidelinkages such alkylphosphonates, phosphorothioates, phosphorodithioates,alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphateesters, carbamates, acetamidate, carboxymethyl esters, carbonates, andphosphate triesters. See Brown, 1994 Meth. Mol. Biol. vol. 20:1-8;Sonveaux, 1994. Meth. Mol. Biol. Vol. 26:1-72; and Uhlmann et al., 1990.Chem. Rev. vol. 90:543-583. Salts, esters, and other pharmaceuticallyacceptable forms of such compounds are also encompassed.

[0198] In a preferred embodiment, a compound can be a peptide,polypeptide, polypeptide analog, amino acid, or amino acid analog. Sucha compound can be synthesized manually or by an automated synthesizer.

[0199] A compound can be a member of a library of compounds. In aspecific embodiment, the compound is selected from a combinatoriallibrary of compounds comprising peptoids; random biooligomers;diversomers such as hydantoins, benzodiazepines and dipeptides;vinylogous polypeptides; nonpeptidal peptidomimetics; oligocarbamates;peptidyl phosphonates; peptide nucleic acid libraries; antibodylibraries; carbohydrate libraries; and small organic molecule libraries.In a preferred embodiment, the small organic molecule libraries arelibraries of benzodiazepines, isoprenoids, thiazolidinones,metathiazanones, pyrrolidines, morpholino compounds, or diazepindiones.

[0200] In another embodiment, a compound can have a molecular weightless than about 10,000 grams per mole, less than about 5,000 grams permole, less than about 1,000 grams per mole, less than about 500 gramsper mole, less than about 100 grams per mole, and salts, esters, andother pharmaceutically acceptable forms of such compounds.

[0201] Compounds can be evaluated comprehensively for cytotoxicity. Thecytotoxic effects of the compounds can be studied using cell lines,including for example 293 (kidney), HuH7 (liver), and Hela cells overabout 4, 10, 16, 24, 36 or 72-hour periods. In addition, a number ofprimary cells such as normal fibroblasts and peripheral bloodmononuclear cells (PBMCs) can be grown in the presence of compounds atvarious concentrations for about 4 days. Fresh compound can be addedevery other day to maintain a constant level of exposure with time. Theeffect of each compound on cell-proliferation can be determined byCellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega Co,Madison, Wis.) and [³H]-Thymidine incorporation. Treatment of some cellswith some of the compounds may have cytostatic effects. A selectiveindex (ratios of CC₅₀ in cytotoxicity assays to the EC₅₀ in ELISA orFACS or the reporter gene assays) for each compound can be calculatedfor all of the UTR-reporters and protein inhibition assays. Compoundsexhibiting substantial selective indices can be of interest and can beanalyzed further in the functional assays.

[0202] The structure of a compound can be determined by any well-knownmethod such as mass spectroscopy, NMR, vibrational spectroscopy, orX-ray crystallography as part of a method of the present invention.

[0203] Compounds can be pharmacologic agents already known in the art orcan be compounds previously unknown to have any pharmacologicalactivity. The compounds can be naturally occurring or designed in thelaboratory. They can be isolated from microorganisms, animals, orplants, and can be produced recombinantly, or synthesized by chemicalmethods known in the art. If desired, compounds can be obtained usingany of the numerous combinatorial library methods known in the art,including but not limited to, biological libraries, spatiallyaddressable parallel solid phase or solution phase libraries, syntheticlibrary methods requiring deconvolution, the “one-bead one-compound”library method, and synthetic library methods using affinitychromatography selection. Methods for the synthesis of molecularlibraries are well known in the art (see, for example, DeWitt et al.,Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl.Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al., J. Med. Chem. 37,2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew.Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al., Angew. Chem. Int.Ed. Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994).Libraries of compounds can be presented in solution (see, e.g.,Houghten, BioTechniques 13, 412-421, 1992), or on beads (Lam, Nature354, 82-84, 1991), chips (Fodor, Nature 364, 555-556, 1993), bacteria orspores (Ladner, U.S. Pat. No. 5,223,409), plasmids (Cull et al., Proc.Natl. Acad. Sci. U.S.A. 89, 1865-1869, 1992), or phage (Scott & Smith,Science 249, 386-390, 1990; Devlin, Science 249, 404-406, 1990); Cwirlaet al., Proc. Natl. Acad. Sci. 97, 6378-6382, 1990; Felici, J. Mol.Biol. 222, 301-310, 1991; and Ladner, U.S. Pat. No. 5,223,409).

[0204] Methods of the present invention for screening compounds canselect for compounds capable of modulating gene expression, which arecapable of directly binding to a ribonucleic acid molecule transcribedfrom a target gene. In a preferred embodiment, a compound identified inaccordance with the methods of the present invention may be capable ofbinding to one or more trans-acting factors (such as, but not limitedto, proteins) that modulate UTR-dependent expression of a target gene.In another preferred embodiment, a compound identified in accordancewith the methods of invention may disrupt an interaction between the 5′UTR and the 3′ UTR.

[0205] Compounds can be tested using in vitro assays (e.g., cell-freeassays) or in vivo assays (e.g., cell-based assays) well known to one ofskill in the art or as provided in the present invention. A compoundthat modulates expression of a target gene can be determined from themethods provided in the present invention. A UTR of the presentinvention includes UTRs capable of modulating gene expression in thepresence, in the absence, or in the presence and absence of a compound.In a preferred embodiment, the effect of a compound on the expression ofone or more genes can be determined utilizing assays well known to oneof skill in the art or provided by the present invention to assess thespecificity of a particular compound's effect on the UTR-dependentexpression of a target gene. In a more preferred embodiment, a compoundhas specificity for a plurality of genes. In another more preferredembodiment, a compound identified utilizing the methods of the presentinvention is capable of specifically effect the expression of only onegene or, alternatively, a group of genes within the same signalingpathway. Compounds identified in the assays of the present invention canbe tested for biological activity using host cells containing orengineered to contain the target RNA element involved in UTR-dependentgene expression coupled to a functional readout system.

[0206] Screening Assays

[0207] The present invention includes and provides for assays capable ofscreening for compounds capable of modulating gene expression. In apreferred aspect of the present invention, an assay is an in vitroassay. In another aspect of the present invention, an assay is an invivo assay. In another preferred aspect of the present invention, anassay measures translation. In a preferred aspect of the presentinvention, the assay includes a nucleic acid molecule of the presentinvention or a construct of the present invention. A nucleic acidmolecule or construct of the present invention include, withoutlimitation, SEQ ID NOs: 3-12, or a sequence that differs from any of theresidues in SEQ ID NOs: 3-12 in that the nucleic acid sequence has beendeleted, substituted, or added in a manner that does not alter thefunction. The present invention also provides fragments and complementsof all the nucleic acid molecules of the present invention.

[0208] In one embodiment of the present invention, the activity orexpression of a reporter gene is modulated. Modulated means increased ordecreased during any point before, after, or during translation. In apreferred embodiment, activity or expression of a reporter gene ismodulated during translation. For example, inhibition of translation ofthe reporter gene would modulate expression. In an alternative example,expression level of a reporter gene is modulated if the steady-statelevel of the expressed protein decreased even though translation was notinhibited. For example, a change in the half-life of a mRNA can modulateexpression.

[0209] In an alternative embodiment, modulated activity or expression ofa reporter gene means increased or decreased during any point before orduring translation.

[0210] In a more preferred aspect, the activity or expression of areporter gene or a target gene is modulated by greater than 30%, 40%,50%, 60%, 70%, 80% or 90% in the presence of a compound. In a highlypreferred aspect, more of an effect is observed in VEGF-dependent cancercells.

[0211] In a most preferred aspect, the activity or expression of areporter gene is modulated without altering the activity of a controlgene for general, indiscriminate translation activity. As used herein,indiscriminate translation activity refers to modulation in translationlevels or activity that is random or unsystematic. One assay formodulation in general, indiscriminate translation activity uses ageneral translational inhibitor, for example puromycin, which is aninhibitor that causes release of nascent peptide and mRNA fromribosomes.

[0212] Expression of a reporter gene can be detected with, for example,techniques know in the art. Translation or transcription of a reportergene can be detected in vitro or in vivo. In detection assays, eitherthe compound or the reporter gene can comprise a detectable label, suchas a fluorescent, radioisotopic, chemiluminescent, or enzymatic label,such as horseradish peroxidase, alkaline phosphatase, or luciferase.

[0213] High-throughput screening can be done by exposing nucleic acidmolecules of the present invention to a library of compounds anddetecting gene expression with assays known in the art, including, forexample without limitation, those described above. In one embodiment ofthe present invention, cancer cells, such as HeLa cells, expressing anucleic acid molecule of the present invention are treated with alibrary of compounds. Percent inhibition of reporter gene activity canbe obtained with all the library compounds can be analyzed using, forexample without limitation, a scattergram generated by SpotFire®(SpotFire, Inc., Somerville, Mass.). The high-throughput screen can befollowed by subsequent selectivity screens. In a preferred embodiment, asubsequent selectivity screen can include detection of reporter geneexpression in cells expressing, for example, a reporter gene linked to aPTCRE or flanked by a 5′ and 3′ UTR of the same gene, either of whichcontains a PTCRE or a NeRP or both of the present invention. In analternative preferred embodiment, a subsequent selectivity screen caninclude detection of reporter gene expression in cells in the presenceof a various concentrations of compounds.

[0214] Once a compound has been identified to modulate UTR-dependentexpression of a target gene and preferably, the structure of thecompound has been identified by the methods described in the presentinvention and well known in the art, the compounds are tested forbiological activity in further assays and/or animal models. Further, alead compound may be used to design congeners or analogs.

[0215] A wide variety of labels and conjugation techniques are known bythose skilled in the art and can be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to PTCREs or NeRPs of the presentinvention include oligolabeling, nick translation, end-labeling, or PCRamplification using a labeled nucleotide. Suitable reporter molecules orlabels which can be used for ease of detection include radionuclides,enzymes, and fluorescent, chemiluminescent, or chromogenic agents, aswell as substrates, cofactors, inhibitors, magnetic particles, and thelike.

[0216] In Vitro

[0217] The present invention includes and provides for assays capable ofscreening for compounds capable of modulating gene expression. In apreferred aspect of the present invention, an assay is an in vitroassay. In a preferred aspect of the present invention, an in vitro assaythat measures translation. In a preferred aspect of the presentinvention the in vitro assay includes a nucleic acid molecule of thepresent invention or a construct of the present invention.

[0218] In one embodiment, a reporter gene of the present invention canencode a fusion protein or a fusion protein comprising a domain thatallows the expressed reporter gene to be bound to a solid support. Forexample, glutathione-S-transferase fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, which are then combined withthe compound or the compound and the non-adsorbed expressed reportergene; the mixture is then incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads or microtiter plate wells are washed toremove any unbound components. Binding of the interactants can bedetermined either directly or indirectly, as described above.Alternatively, the complexes can be dissociated from the solid supportbefore binding is determined.

[0219] Other techniques for immobilizing an expressed reporter gene orcompound on a solid support also can be used in the screening assays ofthe invention. For example, either an expressed reporter gene orcompound can be immobilized utilizing conjugation of biotin andstreptavidin. Biotinylated expressed reporter genes or compounds can beprepared from biotin-NHS(N-hydroxysuccinimide) using techniques wellknown in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,Ill.) and immobilized in the wells of streptavidin-coated 96 well plates(Pierce Chemicals, Rockford, Ill.). Alternatively, antibodies whichspecifically bind to an expressed reporter gene or compound, but whichdo not interfere with a desired binding or catalytic site, can bederivatized to the wells of the plate. Unbound target or protein can betrapped in the wells by antibody conjugation.

[0220] Methods for detecting such complexes, in addition to thosedescribed above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies which specifically bind toan expressed reporter gene or compound, enzyme-linked assays which relyon detecting an activity of an expressed reporter gene, electrophoreticmobility shift assays (EMSA), and SDS gel electrophoresis under reducingor non-reducing conditions.

[0221] In one embodiment, translation of a reporter gene in vitro can bedetected following the use of a reticulocyte lysate translation system,for example the TnT® Coupled Reticulocyte Lysate System (Promega Co.,Madison, Wis.). In this aspect, for example, without limitation, RNA(100 ng) can be translated at 30° C. in reaction mixtures containing 70%reticulocyte lysate, 20 μM amino acids and RNase inhibitor (0.8units/μl). After 45 minutes of incubation, 20 μl of Luclite can be addedand luminescence can be read on the View-Lux. Different concentrationsof compounds can be added to the reaction in a final DMSO concentrationof 2% and the EC₅₀ values calculated. Puromycin can be used as controlfor general indiscriminate translation inhibition. In vitro transcriptsencoding a reporter gene linked to specific UTRs from target genes,including GAPDH, XIAP, TNF-α, and HIF-1α, can also be used.

[0222] To study the influence of cell-type specific factors, capped RNAcan be translated in translation extracts prepared from specializedcells or cancer cell lines, for example without limitation, HT1080 cells(a human fibrosarcoma cell line). Briefly, the cells can be washed withPBS and swollen in hypotonic buffer (10 mM Hepes, pH 7.4, 15 mM KCl, 1.5mM Mg(OAc)₂, 2 mM DTT and 0.5 mM Pefabloc (Pentapharm Ltd. Co.,Switzerland) for 5 minutes on ice. The cells can be lysed using a Douncehomogenizer (100 strokes), and the extracts can be spun for 10 minutesat 10,000×g. These clarified extracts can then be flash-frozen in liquidnitrogen and stored in aliquots at −70° C. The translation reaction canbe capped RNA (50 ng) in a reaction mixture containing 60% clarifiedtranslation extract, 15 μM total amino acids, 0.2 mg/ml Creatinephosho-kinase, which are all in 1× translation buffer (15 mM Hepes, pH7.4, 85 mM KOAc, 1.5 mM Mg(OAc)₂, 0.5 mM ATP, 0.075 mM GTP, 18 mMcreatine diphosphate and 1.5 mM DTT). After incubation of thetranslation reaction for 90 min at 37° C., activity of the proteinencoded by the reporter gene can be detected. For activity ofluciferase, encoded by the luciferase gene serving as the reporter gene,addition of 20 μl of LucLite® (Packard Instrument Co., Inc., Meriden,Conn.) can be used.

[0223] Capped and uncapped RNAs can be synthesized in vitro using the T7polymerase transcription kits (Ambion Inc., Austin, Tex.). Capped RNAsfrom a variety of nucleic acid molecules of the present invention,including without limitation, constructs with VEGF linked to a PTCRE ofthe present invention, constructs with a reporter gene linked only to avector, constructs with GAPDH linked to a PTCRE, constructs with aHIF-1α linked to a PTCRE, and constructs with a HIF-1α not linked to aPTCRE, can be used in a similar in vitro system to study the influenceof cell-type specific factors on translation.

[0224] In Vivo

[0225] The present invention includes and provides for assays capable ofscreening for compounds capable of modulating gene expression. In apreferred aspect of the present invention, an assay is an in vivo assay.A preferred aspect of the present invention is an assay that measurestranslation. In a preferred embodiment of the present invention, an invivo assay includes a nucleic acid molecule of the present invention ora construct of the present invention and can include the use of a cellor a cell or tissue within an organism. In a more preferred embodiment,an in vivo assay includes a nucleic acid molecule of the presentinvention present in a cell or a cell or tissue within an organism.

[0226] In another embodiment, in vivo translation of a reporter gene canbe detected. In a preferred embodiment, a reporter gene is transfectedinto a cancer cell obtained from a cell line available at the (AmericanType Culture Collection (ATCC), Manassas, Va.), for example HeLa, MCF-7,and COS-7, BT474. In a more preferred embodiment, a cancer cell has analtered genome relative to a similarly derived normal, primary cell, andthe mammalian cancer cell proliferates under conditions where such aprimary cell would not.

[0227] Screening for compounds that modulate reporter gene expressioncan be carried out in an intact cell. Any cell that comprises a reportergene can be used in a cell-based assay system. A reporter gene can benaturally occurring in the cell or can be introduced using techniquessuch as those described above (see Cells and Organisms). In oneembodiment, a cell line is chosen based on its expression levels ofnaturally occurring VEGF. Modulation of reporter gene expression by acompound can be determined in vitro as described above or in vivo asdescribed below.

[0228] To detect expression of endogenous protein, a variety ofprotocols for detecting and measuring the expression of a reporter geneare known in the art. For example, Enzyme-Linked Immunosorbent Assays(ELISAs), western blots using either polyclonal or monoclonal antibodiesspecific for an expressed reporter gene, Fluorescence-Activated CellSorter (FACS), electrophoretic mobility shift assays (EMSA), orradioimmunoassay (RIA) can be performed to quantify the level ofspecific proteins in lysates or media derived from cells treated withthe compounds. In a preferred embodiment, a phenotypic or physiologicalreadout can be used to assess UTR-dependent activity of the target RNAin the presence and absence of the lead compound.

[0229] A wide variety of labels and conjugation techniques are known bythose skilled in the art and can be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides having a PTCREor a NeRP of the present invention include oligolabeling, nicktranslation, end-labeling, or PCR amplification using a labelednucleotide. Alternatively, sequences having a PTCRE or a NeRP of thepresent invention can be cloned into a vector for the production of amRNA probe. Such vectors are known in the art, are commerciallyavailable, and can be used to synthesize RNA probes in vitro by additionof labeled nucleotides and an appropriate RNA polymerase such as T7, T3,or SP6. These procedures can be conducted using a variety ofcommercially available kits (Amersham Biosciences Inc., Piscataway,N.J.; and Promega Co, Madison, Wis.). Suitable reporter molecules orlabels which can be used for ease of detection include radionucleotides,enzymes, and fluorescent, chemiluminescent, or chromogenic agents, aswell as substrates, cofactors, inhibitors, magnetic particles, and thelike.

[0230] Therapeutic Uses

[0231] The present invention also provides for methods for treating,preventing or ameliorating one or more symptoms of a disease or disorderassociated with the aberrant expression of a target gene, said methodcomprising administering to a subject in need thereof a therapeuticallyor prophylactically effective amount of a compound, or apharmaceutically acceptable salt thereof, identified according to themethods described herein. In one embodiment of the present invention, atarget gene is aberrantly expressed. A target gene can be aberrantlyoverexpressed or expressed at an aberrantly low level. In particular,the invention provides for a method of treating or preventing a diseaseor disorder or ameliorating a symptom thereof, said method comprisingadministering to a subject in need thereof an effective amount of acompound, or a pharmaceutically acceptable salt thereof, identifiedaccording to the methods described herein, wherein said effective amountincreases the expression of a target gene beneficial in the treatment orprevention of said disease or disorder. The invention also provides fora method of treating or preventing a disease or disorder or amelioratinga symptom thereof, said method comprising administering to a subject inneed thereof an effective amount of a compound, or a pharmaceuticallyacceptable salt thereof, identified according to the methods describedherein, wherein said effective amount decreases the expression of atarget gene whose expression is associated with or has been linked tothe onset, development, progression or severity of said disease ordisorder. In a specific embodiment, the disease or disorder is aproliferative disorder, an inflammatory disorder, an infectious disease,a genetic disorder, an autoimmune disorder, a cardiovascular disease, ora central nervous system disorder. In an embodiment wherein the diseaseor disorder is an infectious disease, the infectious disease can becaused by a fungal infection, a bacterial infection, a viral infection,or an infection caused by another type of pathogen.

[0232] In addition, the invention provides pharmaceutical compositionsthat can be administered to a patient to achieve a therapeutic effect.Pharmaceutical compositions of the invention can comprise, for example,ribozymes or antisense oligonucleotides, antibodies that specificallybind to a PTCRE or NeRP of the present invention, or mimetics,activators, inhibitors of PTCRE or NeRP activity, or a nucleic acidmolecule of the present invention. The compositions can be administeredalone or in combination with at least one other agent, such asstabilizing compound, which can be administered in any sterile,biocompatible pharmaceutical carrier, including, but not limited to,saline, buffered saline, dextrose, and water. The compositions can beadministered to a patient alone, or in combination with other agents,drugs or hormones.

[0233] In addition to the active ingredients, these pharmaceuticalcompositions can contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Pharmaceutical compositions of the invention can be administered by anynumber of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, subcutaneous, intraperitoneal,intranasal, parenteral, topical, sublingual, or rectal means.Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

[0234] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents can be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0235] Pharmaceutical preparations that can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with fillers or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds can be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0236] Pharmaceutical formulations suitable for parenteraladministration can be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions can contain substances that increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds can beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Non-lipid polycationic amino polymers also can be used for delivery.Optionally, the suspension also can contain suitable stabilizers oragents that increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions. For topical or nasaladministration, penetrants appropriate to the particular barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

[0237] The pharmaceutical compositions of the present invention can bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes. Thepharmaceutical composition can be provided as a salt and can be formedwith many acids, including but not limited to, hydrochloric, sulfuric,acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be moresoluble in aqueous or other protonic solvents than are the correspondingfree base forms. In other cases, the preferred preparation can be alyophilized powder which can contain any or all of the following: 1-50mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5to 5.5, that is combined with buffer prior to use. Further details ontechniques for formulation and administration can be found in the latestedition of Remington's Pharmaceutical Sciences (Maack Publishing Co.,Easton, Pa.). After pharmaceutical compositions have been prepared, theycan be placed in an appropriate container and labeled for treatment ofan indicated condition. Such labeling would include amount, frequency,and method of administration.

[0238] Determination of a Therapeutically Effective Dose

[0239] A therapeutically effective dose refers to that amount of activeingredient that increases or decreases reporter gene activity relativeto reporter gene activity that occurs in the absence of thetherapeutically effective dose. For any compound, the therapeuticallyeffective dose can be estimated initially either in cell culture assaysor in animal models, usually mice, rabbits, dog, or pigs. The animalmodel also can be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0240] Therapeutic efficacy and toxicity, e.g., ED₅₀ (the dosetherapeutically effective in 50% of the population) and LD₅₀ (the doselethal to 50% of the population), can be determined by standardpharmaceutical procedures in cell cultures or experimental animals. Thedose ratio of toxic to therapeutic effects is the therapeutic index, andit can be expressed as the ratio, LD₅₀/ED₅₀.

[0241] Pharmaceutical compositions that exhibit large therapeuticindices are preferred. The data obtained from cell culture assays andanimal studies is used in formulating a range of dosage for human use.The dosage contained in such compositions is preferably within a rangeof circulating concentrations that include the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0242] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activeingredient or to maintain the desired effect. Factors that can be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions can be administered every 3 to 4 days, everyweek, or once every two weeks depending on the half-life and clearancerate of the particular formulation.

[0243] Normal dosage amounts can vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0244] If the reagent is a single-chain antibody, polynucleotidesencoding the antibody can be constructed and introduced into a celleither ex vivo or in vivo using well-established techniques including,but not limited to, transferrin-polycation-mediated DNA transfer,transfection with naked or encapsulated nucleic acids, liposome-mediatedcellular fusion, intracellular transportation of DNA-coated latex beads,protoplast fusion, viral infection, electroporation, “gene gun,” andDEAE- or calcium phosphate-mediated transfection.

[0245] Effective in vivo dosages of an antibody are in the range ofabout 5 μg to about 50 μg/kg, about 50 μg to about 5 mg/kg, about 100 μgto about 500 μg/kg of patient body weight, and about 200 to about 250μg/kg of patient body weight. For administration of polynucleotidesencoding single-chain antibodies, effective in vivo dosages are in therange of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 μgto about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100μg of DNA.

[0246] If the expression product is mRNA, the reagent is preferably anantisense oligonucleotide or a ribozyme. Polynucleotides that expressantisense oligonucleotides or ribozymes can be introduced into cells bya variety of methods, as described above.

[0247] Preferably, a reagent reduces expression of a reporter gene orthe activity of a reporter gene by at least about 10, preferably about50, more preferably about 75, 90, or 100% relative to the absence of thereagent. The effectiveness of the mechanism chosen to decrease the levelof expression of a reporter gene or the activity of a reporter gene canbe assessed using methods well known in the art, such as hybridizationof nucleotide probes to reporter gene-specific mRNA, quantitativeRT-PCR, immunologic detection of an expressed reporter gene, ormeasurement of activity from an expressed reporter gene.

[0248] In any of the embodiments described above, any of thepharmaceutical compositions of the invention can be administered incombination with other appropriate therapeutic agents. Selection of theappropriate agents for use in combination therapy can be made by one ofordinary skill in the art, according to conventional pharmaceuticalprinciples. The combination of therapeutic agents can actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

[0249] Any of the therapeutic methods described above can be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0250] Administration of a Therapeutically Effective Dose

[0251] A reagent which affects translation can be administered to ahuman cell, either in vitro or in vivo, to specifically reducetranslational activity of a specific gene. In a preferred embodiment,the reagent preferably binds to a 5′ UTR of a gene. In an alternateembodiment, the present invention the reagent preferably binds to aPTCRE or NeRP of the present invention. In a preferred embodiment, thereagent is a compound. For treatment of human cells ex vivo, an antibodycan be added to a preparation of stem cells which have been removed fromthe body. The cells can then be replaced in the same or another humanbody, with or without clonal propagation, as is known in the art.

[0252] In one embodiment, the reagent is delivered using a liposome.Preferably, the liposome is stable in the animal into which it has beenadministered for at least about 30 minutes, more preferably for at leastabout 1 hour, and even more preferably for at least about 24 hours. Aliposome comprises a lipid composition that is capable of targeting areagent, particularly a polynucleotide, to a particular site in ananimal, such as a human. Preferably, the lipid composition of theliposome is capable of targeting to a specific organ of an animal, suchas the lung, liver, spleen, heart brain, lymph nodes, and skin.

[0253] A liposome useful in the present invention comprises a lipidcomposition that is capable of fusing with the plasma membrane of thetargeted cell to deliver its contents to the cell. Preferably, thetransfection efficiency of a liposome is about 0.5 μg of DNA per 16nmole of liposome delivered to about 10⁶ cells, more preferably about1.0 μg of DNA per 16 nmole of liposome delivered to about 10⁶ cells, andeven more preferably about 2.0 μg of DNA per 16 nmol of liposomedelivered to about 10⁶ cells. Preferably, a liposome is between about100 and 500 nm, more preferably between about 150 and 450 nm, and evenmore preferably between about 200 and 400 nm in diameter.

[0254] Suitable liposomes for use in the present invention include thoseliposomes standardly used in, for example, gene delivery methods knownto those of skill in the art. More preferred liposomes include liposomeshaving a polycationic lipid composition and/or liposomes having acholesterol backbone conjugated to polyethylene glycol. Optionally, aliposome comprises a compound capable of targeting the liposome to aparticular cell type, such as a cell-specific ligand exposed on theouter surface of the liposome.

[0255] Complexing a liposome with a reagent such as an antisenseoligonucleotide or ribozyme can be achieved using methods that arestandard in the art (see, for example, U.S. Pat. No. 5,705,151).Preferably, from about 0.1 μg to about 10 μg of polynucleotide iscombined with about 8 nmol of liposomes, more preferably from about 0.5μg to about 5 μg of polynucleotides are combined with about 8 nmolliposomes, and even more preferably about 1.0 μg of polynucleotides iscombined with about 8 nmol liposomes.

[0256] In another embodiment, antibodies can be delivered to specifictissues in vivo using receptor-mediated targeted delivery.Receptor-mediated DNA delivery techniques are taught in, for example,Findeis et al. Trends in Biotechnol. 11, 202-05 (1993); Chiou et al.,Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988); Wu etal., J. Biol. Chem. 269, 542-46 (1994); Zenke et al., Proc. Natl. Acad.Sci. U.S.A. 87, 3655-59 (1990); Wu et al., J. Biol. Chem. 266, 338-42(1991).

[0257] Diagnostic Methods

[0258] Agents of the present invention can also be used in diagnosticassays for detecting diseases and abnormalities or susceptibility todiseases and abnormalities related to the presence of mutations in thenucleic acid sequences that encode a PTCRE or NeRP of the presentinvention. For example, differences can be determined between the cDNAor genomic sequence encoding a PTCRE or NeRP in individuals afflictedwith a disease and in normal individuals. If a mutation is observed insome or all of the afflicted individuals but not in normal individuals,then the mutation is likely to be the causative agent of the disease.

[0259] For example, the direct DNA sequencing method can reveal sequencedifferences between a reference gene and a gene having mutations. Inaddition, cloned DNA segments can be employed as probes to detectspecific DNA segments. The sensitivity of this method is greatlyenhanced when combined with PCR. For example, a sequencing primer can beused with a double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures using radiolabeled nucleotides orby automatic sequencing procedures using fluorescent tags.

[0260] Moreover, for example, genetic testing based on DNA sequencedifferences can be carried out by detection of alteration inelectrophoretic mobility of DNA fragments in gels with or withoutdenaturing agents. Small sequence deletions and insertions can bevisualized, for example, by high-resolution gel electrophoresis. DNAfragments of different sequences can be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science 230, 1242, 1985). Sequence changes at specific locationscan also be revealed by nuclease protection assays, such as RNase and S1protection or the chemical cleavage method (e.g., Cotton et al., Proc.Natl. Acad. Sci. USA 85, 4397-4401, 1985). Thus, the detection of aspecific DNA sequence can be performed by methods such as hybridization,RNase protection, chemical cleavage, direct DNA sequencing or the use ofrestriction enzymes and Southern blotting of genomic DNA. In addition todirect methods such as gel-electrophoresis and DNA sequencing, mutationscan also be detected by in situ analysis.

[0261] Altered levels of a PTCRE or NeRP of the present invention canalso be detected in various tissues. For example, one or more geneshaving a PTCRE or a NeRP can be detected by assays used to detect levelsof particular nucleic acid sequence, such as Southern hybridization,northern hybridization, and PCR. Alternatively, assays can be used todetect levels of a reporter polypeptide regulated by a PTCRE or a NeRPor of a polypeptide encoded by a gene having a PTCRE or a NeRP. Suchassays are well known to those of skill in the art and includeradioimmunoassays, competitive binding assays, western blot analysis,and ELISA assays. A sample from a subject, such as blood or a tissuebiopsy derived from a host, may be the material on which these assaysare conducted.

[0262] Having now generally described the invention, the same will bemore readily understood through reference to the following examples thatare provided by way of illustration, and are not intended to be limitingof the present invention, unless specified.

[0263] Each periodical, patent, and other document or reference citedherein is herein incorporated by reference in its entirety.

EXAMPLES Example 1 Identification of Compounds that Specifically InhibitVEGF Expression Post-transcriptionally

[0264] A monocistronic reporter construct (pLuc/vegf5′+3′ UTR) is underthe transcriptional control of the CMV promoter and contains a VEGF IRESdriving the luciferase reporter, which nucleic acid sequences are bothupstream of a VEGF 3′-UTR. Stable cell lines are generated bytransfecting 293 cells with the pLuc/vegf5′+3′ UTR. A stable cell lineis cultured under hygromycin B selection to create clonal cell linesconsistent with protocols well known in the art. After two weeks ofselection, clonal cell lines are screened for luciferase activity. Theluciferase activity of several clonal cell lines (hereafter “clones”)are compared and normalized against total protein content. Clones aremaintained under hygromycin B selection for more than three months withintermittent monitoring of luciferase activity. Clones are stable andmaintain a high level of luciferase expression. Many clones, forexample, about twenty, may be compared to each other with respect toluciferase activity. In comparison to clones B9, D3, and H6, clone B9exhibits the highest level of luciferase activity. In addition,semi-quantitative PCR analysis is performed, and the results indicatethat multiple copies of the reporter are integrated per cell. Particularparameters for clones are studied prior to selection for use inpost-transcriptional, high-throughput screening (PTHTS). Relevantparameters for PTHTS include, but are not limited to, cell number,incubation time, DMSO concentration, and volume of substrate.

[0265] Chemical libraries in excess of 150,000 compounds are screened byPTHTS with a clone containing the monocistronic reporter construct,pLuc/vegf5′+3′ UTR. Screens are performed in duplicate with eachmolecule at a single concentration of 7.5 μM. Bright-Glow (Promega Co.,Madison, Wis.) is used as a substrate to measure firefly luciferaseactivity. Active compounds are identified by reporting the averagepercent inhibition of the duplicate runs followed by rejecting thosecompounds that did not provide satisfactory reproducibility. The averagepercent inhibition of compounds that provide satisfactoryreproducibility is within a range of about 10%, about 25% or about 35%in duplicate runs. Data is analyzed as a normal distribution, which isapparent from graphical and statistical analysis of skewness andkurtosis. Hits are then reported at about a 99% confidence level,usually representing a selection of 3 standard deviations from the mean,or a hit lower limit of observed inhibition about equal to 50%. Theseselection criteria result in a hit rate of about 1%.

[0266] Certain compounds that are identified through the PTHTS-screeningtier by screening with clone B9 modulate hypoxia-inducible endogenousVEGF expression. Endogenous VEGF protein levels are monitored by anELISA assay (R&D Systems, Minneapolis, Minn.). HeLa cells are used toevaluate hypoxia-inducible expression. HeLa cells demonstrate about athree- to five-fold hypoxia-inducible window as compared to normoxicconditions (about 1000—about 1500 pg/ml under hypoxia compared to about200—about 400 pg/ml under normoxia). Cells are cultured overnight underhypoxic conditions (about 1% O₂, about 5% CO₂, and balanced withnitrogen) in the presence or absence of compounds. The conditioned mediais assayed by ELISA. The concentration of VEGF is calculated from thestandard ELISA curve of each assay. The assays are performed induplicate at a compound concentration of about 7 μM. A threshold ofabout 50% inhibition for a compound is selected as a criterion forfurther investigation. Further evaluation of about 100 to about 150compounds is conducted from about 700 to about 800 initial PTHTS hits.The activity of the identified compounds is confirmed by repeating theexperiments described above. The identified compounds are then acquiredas dry powders and analyzed further. The purity and molecular weight ofthe identified compounds are confirmed by LC-MS.

[0267] A dose-response analysis is performed using an ELISA assay andusing conditions essentially as described above. A series of sevendifferent concentrations are analyzed. In parallel, a dose-responsecytotoxicity assay is performed under the same conditions as the ELISAto ensure that the inhibition of VEGF expression is not due tocytotoxicity as measured by CellTiter-Glo® (Promega, Inc., Madison,Wis.). Dose-response curves are plotted using percentage inhibitionversus concentration of the compound.

[0268] For each compound, the maximal inhibition is set as 100% and theminimal inhibition is set as 0% to generate EC₅₀ and CC₅₀ values. Acompound from PTHTS shows a sigmoidal curve over a compoundconcentration range from about 10⁻¹ nM to about 10⁴ nM when plottedagainst the percent inhibition of VEGF expression on the y-axis (seeFIG. 2). The same compound from PTHTS shows a convex curve over the samecompound concentration range plotted against the percent ofcytotoxicity. The ELISA EC₅₀ (50% inhibition of VEGF expression) forthis 1234particular compound is about 7 nM, while its CC₅₀ (50%cytotoxicity) is greater than about 2000 nN. Subsets of compounds thatshow similar efficacy/cytotoxicity windows are also identified.

[0269] The B9 cell line harbors the firefly luciferase reporter drivenby the CMV promoter and flanked by the 5′- and 3′-UTRs of VEGFtranscripts. Use of the B9 cell line with the PTHTS identifies compoundsthat specifically target the function of VEGF UTRs to modulateexpression. Cell line B12 harbors the luciferase reporter in the absenceof operably linked VEGF UTRs. Compounds that inhibit luciferase activityin both the B9 and B12 cell lines are general transcription or generaltranslation inhibitors or luciferase enzyme inhibitors. Several UTRspecific compounds are identified in experiments with PTHTS identifiedcompounds as described above. The dose-response curves of an identifiedcompound show a concave curve in B9 cells and a sigmoidal curve in B12cells when the percent luciferase inhibition of each is plotted over acompound concentration range from about 10⁻¹ nM to about 10⁴ nM on thex-axis (see FIG. 3). The difference between the two cell lines (B9 andB12) shows that inhibition of VEGF production by this compound isthrough the VEGF UTRs, i.e., by a post-transcriptional controlmechanism. A control experiment is performed with a general translationinhibitor, puromycin. Puromycin treatment does not change the differenceof inhibition in luciferase expression in these two cell lines.

Example 2 Characteristics of UTR-specific VEGF Inhibitors

[0270] All identified compounds are re-synthesized and shown by LC/MSand combustion analysis to be greater than 95% pure. Subsequently, there-synthesized compounds are tested in the dose-response VEGF ELISA andluciferase assays that are used to initially assess UTR specificity. Allidentified compounds retain UTR specificity and are bonafide inhibitorsof VEGF expression.

[0271] PTHTS using B9 cells identified compounds that specificallyinhibit hypoxia inducible VEGF expression for the treatment of ocularneovascular diseases. Compounds that target multiple angiogenesisfactors (including VEGF) for the treatment of cancers are alsoidentifiable. Several targets are used for these purposes, includingTNF-α, FGF-2, G-CSF, IGF-1, PDGF, and HIF-1α.

[0272] ELISA assays analyze levels of expression of these factors usingcommercially available kits from R&D Systems (Minneapolis, Minn.).UTR-specific PTHTS identified compounds are tested for their ability toinhibit the expression of a subset of these proteins, including G-CSF,TNFα, FGF-2, and IGF-1. Identified compounds that are very potentinhibitors of VEGF production as assayed in HeLa cells have EC₅₀ valuesranging from low nM to high nM. Treatment with a general translationinhibitor (puromycin) results in similar inhibition for all thesecytokines, with EC₅₀ values ranging from about 0.2 to about 2 μM.

[0273] Lead compounds are further characterized and optimized. Analogsare synthesized and identified compounds exhibit excellent potency inthe VEGF ELISA assay (EC₅₀ values ranging from 0.5 nM to 50 nM). Inanother embodiment, an analog exhibits low nM potency. In an additionalembodiment, several analogs are synthesized and a subset of identifiedcompounds are very active (EC₅₀ values ranging from 1 to 50 nM) in theVEGF ELISA assay. Activity of a very potent analog is improved about500-fold compared to its parent (EC₅₀ of 1 nM vs. 500 nM). Furthercharacterization and optimization for selectivity and pharmaceuticalproperties (ADMET) of the most active compounds will develop a drugcandidate(s) for clinical trials.

Example 3 Identified Compounds are Active as Inhibitors ofHypoxia-inducible VEGF Production in Retinal Pigment Epithelial Cellsand Macrophage Cells

[0274] PTHTS identified compounds are VEGF-specific inhibitors for thetreatment of ocular neovascular disorders. The effect of the identifiedcompounds on retinal pigment epithelial cells and macrophage cells aretested in two cell lines: ARPE-19, a human retinal pigment epithelialcell line, and RAW264, a mouse macrophage cell line. Both cell linesproduce high levels of VEGF under hypoxic conditions. A subset ofidentified compounds is active in these two cell lines. Compound 1inhibits VEGF production in both macrophage (a non-limiting example ofwhich is RAW264.7) and retinal pigment epithelial cells (a non-limitingexample of which is ARPE-19). In selectivity studies, as shown in Table2, Compound 1 specifically inhibits VEGF expression relative to that ofother factors (FGF-2, IGF-1, GCSF, TNFα). TABLE 2 Selectivity studiesCompound 1 Compound 2 Compound 3 ELISA (EC_(50 g) μM) VEGF 0.007-0.020.1-0.5 0.2-1 TNFα >30 >30 >30 G-CSF >30 >30 >30 FGF-2 0.29 >30 >30IGF-1 >30 >30 >30

Example 4 Identified Compounds are Active in Inhibition of VEGFExpression and Tumor Growth In Vivo

[0275] A pharmacodynamic model assesses intratumor VEGF levels andselects compounds for in vivo efficacy. Preliminary data demonstratesthat several of our compounds effectively inhibit VEGF production intumor tissues (see FIG. 4A). Briefly, HT1080 cells (a human fibrosarcomacell line) are implanted subcutaneously in nude mice. After seven days,mice are administrated compounds orally at 20mg/kg/day for two weeks.The tumors are then excised from the mice and homogenized in Tris-HClbuffer containing proteinase inhibitors (Moulder, S. L., et al., CancerRes. 61(24):8887-95, 2001). Intratumor VEGF levels are subsequentlymeasured using a human VEGF ELISA kit (R&D System, Minneapolis, Minn.).Protein concentrations of the homogenates are measured with a Bio-Rad™Protein assay kit and intratumor VEGF levels are normalized to theprotein concentrations. Treatment with the identified compoundssignificantly reduces intratumor VEGF protein levels compared to thevehicle control. In addition, treatment with the identified compound fortwo weeks inhibits tumor growth as compared to the vehicle-treatedcontrol groups (see FIG. 4B).

Example 5 Mapping of Functional Domains in a 5′ VEGF UTR

[0276] VEGF-5UTR1 and 5UTR2 are amplified from human genomic DNA. Thefull-length 5′ UTR is generated by ligation of the two fragments (seeFIG. 5). P2luc/VEGF5UTR-FL is generated by inserting the full-length 5′UTR into a dicistronic plasmid (p2luc) between SalI and SmalI sites.Other vectors are derived from p2luc/VEGF5UTR-FL by deleting relevantsequences. All these plasmids are tested in 293 cells by transienttransfection. FIG. 6 shows the relative fire-fly luciferase activity(normalized against Renilla luciferase) for each of the VEGF 5′ UTRfragments. Similar results are obtained from repeating such experiments.

1 26 1 1038 DNA Artificial Synthetic Construct 1 tcgcggaggc ttggggcagccgggtagctc ggaggtcgtg gcgctggggg ctagcaccag 60 cgctctgtcg ggaggcgcagcggttaggtg gaccggtcag cggactcacc ggccagggcg 120 ctcggtgctg gaatttgatattcattgatc cgggttttat ccctcttctt ttttcttaaa 180 catttttttt taaaactgtattgtttctcg ttttaattta tttttgcttg ccattcccca 240 cttgaatcgg gccgacggcttggggagatt gctctacttc cccaaatcac tgtggatttt 300 ggaaaccagc agaaagaggaaagaggtagc aagagctcca gagagaagtc gaggaagaga 360 gagacggggt cagagagagcgcgcgggcgt gcgagcagcg aaagcgacag gggcaaagtg 420 agtgacctgc ttttgggggtgaccgccgga gcgcggcgtg agccctcccc cttgggatcc 480 cgcagctgac cagtcgcgctgacggacaga cagacagaca ccgcccccag ccccagctac 540 cacctcctcc ccggccggcggcggacagtg gacgcggcgg cgagccgcgg gcaggggccg 600 gagcccgcgc ccggaggcggggtggagggg gtcggggctc gcggcgtcgc actgaaactt 660 ttcgtccaac ttctgggctgttctcgcttc ggaggagccg tggtccgcgc gggggaagcc 720 gagccgagcg gagccgcgagaagtgctagc tcgggccggg aggagccgca gccggaggag 780 ggggaggagg aagaagagaaggaagaggag agggggccgc agtggcgact cggcgctcgg 840 aagccgggct catggacgggtgaggcggcg gtgtgcgcag acagtgctcc agccgcgcgc 900 gctccccagg ccctggcccgggcctcgggc cggggaggaa gagtagctcg ccgaggcgcc 960 gaggagagcg ggccgccccacagcccgagc cggagaggga gcgcgagccg cgccggcccc 1020 ggtcgggcct ccgaaacc1038 2 576 DNA Artificial Synthetic Construct 2 atgaactttc tgctgtcttgggtgcattgg agccttgcct tgctgctcta cctccaccat 60 gccaagtggt cccaggctgcacccatggca gaaggaggag ggcagaatca tcacgaagtg 120 gtgaagttca tggatgtctatcagcgcagc tactgccatc caatcgagac cctggtggac 180 atcttccagg agtaccctgatgagatcgag tacatcttca agccatcctg tgtgcccctg 240 atgcgatgcg ggggctgctgcaatgacgag ggcctggagt gtgtgcccac tgaggagtcc 300 aacatcacca tgcagattatgcggatcaaa cctcaccaag gccagcacat aggagagatg 360 agcttcctac agcacaacaaatgtgaatgc agaccaaaga aagatagagc aagacaagaa 420 aatccctgtg ggccttgctcagagcggaga aagcatttgt ttgtacaaga tccgcagacg 480 tgtaaatgtt cctgcaaaaacacagactcg cgttgcaagg cgaggcagct tgagttaaac 540 gaacgtactt gcagatgtgacaagccgagg cggtga 576 3 702 DNA Artificial Synthetic Construct 3tccagagaga agtcgaggaa gagagagacg gggtcagaga gagcgcgcgg gcgtgcgagc 60agcgaaagcg acaggggcaa agtgagtgac ctgcttttgg gggtgaccgc cggagcgcgg 120cgtgagccct cccccttggg atcccgcagc tgaccagtcg cgctgacgga cagacagaca 180gacaccgccc ccagccccag ctaccacctc ctccccggcc ggcggcggac agtggacgcg 240gcggcgagcc gcgggcaggg gccggagccc gcgcccggag gcggggtgga gggggtcggg 300gctcgcggcg tcgcactgaa acttttcgtc caacttctgg gctgttctcg cttcggagga 360gccgtggtcc gcgcggggga agccgagccg agcggagccg cgagaagtgc tagctcgggc 420cgggaggagc cgcagccgga ggagggggag gaggaagaag agaaggaaga ggagaggggg 480ccgcagtggc gactcggcgc tcggaagccg ggctcatgga cgggtgaggc ggcggtgtgc 540gcagacagtg ctccagccgc gcgcgctccc caggccctgg cccgggcctc gggccgggga 600ggaagagtag ctcgccgagg cgccgaggag agcgggccgc cccacagccc gagccggaga 660gggagcgcga gccgcgccgg ccccggtcgg gcctccgaaa cc 702 4 336 DNA ArtificialSynthetic Construct 4 tcgcggaggc ttggggcagc cgggtagctc ggaggtcgtggcgctggggg ctagcaccag 60 cgctctgtcg ggaggcgcag cggttaggtg gaccggtcagcggactcacc ggccagggcg 120 ctcggtgctg gaatttgata ttcattgatc cgggttttatccctcttctt ttttcttaaa 180 catttttttt taaaactgta ttgtttctcg ttttaatttatttttgcttg ccattcccca 240 cttgaatcgg gccgacggct tggggagatt gctctacttccccaaatcac tgtggatttt 300 ggaaaccagc agaaagagga aagaggtagc aagagc 336 5485 DNA Artificial Synthetic Construct 5 gccggcggcg gacagtggacgcggcggcga gccgcgggca ggggccggag cccgcgcccg 60 gaggcggggt ggagggggtcggggctcgcg gcgtcgcact gaaacttttc gtccaacttc 120 tgggctgttc tcgcttcggaggagccgtgg tccgcgcggg ggaagccgag ccgagcggag 180 ccgcgagaag tgctagctcgggccgggagg agccgcagcc ggaggagggg gaggaggaag 240 aagagaagga agaggagagggggccgcagt ggcgactcgg cgctcggaag ccgggctcat 300 ggacgggtga ggcggcggtgtgcgcagaca gtgctccagc cgcgcgcgct ccccaggccc 360 tggcccgggc ctcgggccggggaggaagag tagctcgccg aggcgccgag gagagcgggc 420 cgccccacag cccgagccggagagggagcg cgagccgcgc cggccccggt cgggcctccg 480 aaacc 485 6 556 DNAArtificial Synthetic Construct 6 cagctgacca gtcgcgctga cggacagacagacagacacc gcccccagcc ccagctacca 60 cctcctcccc ggccggcggc ggacagtggacgcggcggcg agccgcgggc aggggccgga 120 gcccgcgccc ggaggcgggg tggagggggtcggggctcgc ggcgtcgcac tgaaactttt 180 cgtccaactt ctgggctgtt ctcgcttcggaggagccgtg gtccgcgcgg gggaagccga 240 gccgagcgga gccgcgagaa gtgctagctcgggccgggag gagccgcagc cggaggaggg 300 ggaggaggaa gaagagaagg aagaggagagggggccgcag tggcgactcg gcgctcggaa 360 gccgggctca tggacgggtg aggcggcggtgtgcgcagac agtgctccag ccgcgcgcgc 420 tccccaggcc ctggcccggg cctcgggccggggaggaaga gtagctcgcc gaggcgccga 480 ggagagcggg ccgccccaca gcccgagccggagagggagc gcgagccgcg ccggccccgg 540 tcgggcctcc gaaacc 556 7 294 DNAArtificial Synthetic Construct 7 gctagctcgg gccgggagga gccgcagccggaggaggggg aggaggaaga agagaaggaa 60 gaggagaggg ggccgcagtg gcgactcggcgctcggaagc cgggctcatg gacgggtgag 120 gcggcggtgt gcgcagacag tgctccagccgcgcgcgctc cccaggccct ggcccgggcc 180 tcgggccggg gaggaagagt agctcgccgaggcgccgagg agagcgggcc gccccacagc 240 ccgagccgga gagggagcgc gagccgcgccggccccggtc gggcctccga aacc 294 8 194 DNA Artificial Synthetic Construct8 cgggctcatg gacgggtgag gcggcggtgt gcgcagacag tgctccagcc gcgcgcgctc 60cccaggccct ggcccgggcc tcgggccggg gaggaagagt agctcgccga ggcgccgagg 120agagcgggcc gccccacagc ccgagccgga gagggagcgc gagccgcgcc ggccccggtc 180gggcctccga aacc 194 9 476 DNA Artificial Synthetic Construct 9tcgcggaggc ttggggcagc cgggtagctc ggaggtcgtg gcgctggggg ctagcaccag 60cgctctgtcg ggaggcgcag cggttaggtg gaccggtcag cggactcacc ggccagggcg 120ctcggtgctg gaatttgata ttcattgatc cgggttttat ccctcttctt ttttcttaaa 180catttttttt taaaactgta ttgtttctcg ttttaattta tttttgcttg ccattcccca 240cttgaatcgg gccgacggct tggggagatt gctctacttc cccaaatcac tgtggatttt 300ggaaaccagc agaaagagga aagaggtagc aagagctcca gagagaagtc gaggaagaga 360gagacggggt cagagagagc gcgcgggcgt gcgagcagcg aaagcgacag gggcaaagtg 420agtgacctgc ttttgggggt gaccgccgga gcgcggcgtg agccctcccc cttggg 476 10 554DNA Artificial Synthetic Construct 10 tcgcggaggc ttggggcagc cgggtagctcggaggtcgtg gcgctggggg ctagcaccag 60 cgctctgtcg ggaggcgcag cggttaggtggaccggtcag cggactcacc ggccagggcg 120 ctcggtgctg gaatttgata ttcattgatccgggttttat ccctcttctt ttttcttaaa 180 catttttttt taaaactgta ttgtttctcgttttaattta tttttgcttg ccattcccca 240 cttgaatcgg gccgacggct tggggagattgctctacttc cccaaatcac tgtggatttt 300 ggaaaccagc agaaagagga aagaggtagcaagagctcca gagagaagtc gaggaagaga 360 gagacggggt cagagagagc gcgcgggcgtgcgagcagcg aaagcgacag gggcaaagtg 420 agtgacctgc ttttgggggt gaccgccggagcgcggcgtg agccctcccc cttgggatcc 480 cgcagctgac cagtcgcgct gacggacagacagacagaca ccgcccccag ccccagctac 540 cacctcctcc ccgg 554 11 51 DNAArtificial Synthetic Construct 11 tcgcggaggc ttggggcagc cgggtagctcggaggtcgtg gcgctggggg c 51 12 91 DNA Artificial Synthetic construct 12tcgcggaggc ttggggcagc cgggtagctc ggaggtcgtg gcgctggggg ctagcaccag 60cgctctgtcg ggaggcgcag cggttaggtg g 91 13 335 DNA Artificial SyntheticConstruct 13 tcgcggaggc ttggggcagc cgggtagctc ggaggtcgtg gcgctgggggctagcaccag 60 cgctctgtcg ggaggcgcag cggttaggtg gaccggtcag cggactcaccggccagggcg 120 ctcggtgctg gaatttgata ttcattgatc cgggttttat ccctcttcttttttcttaaa 180 catttttttt taaaactgta ttgtttctcg ttttaattta tttttgcttgccattcccca 240 cttgaatcgg gccgacggct tggggagatt gctctacttc cccaaatcactgtggatttt 300 ggaaaccagc agaaagagga aagaggtagc aagag 335 14 332 DNAArtificial Synthetic Construct 14 tcgcggaggc ttggggcagc cgggtagctcggaggcgtgg cgctgggggc tagcaccagc 60 gctctgtcgg gaggcgcagc ggttaggtggaccggtcagc ggactcaccg gccagggcgc 120 tcggtgctgg aatttgatat tcattgatccgggttttatc cctcttcttt tttcttaaac 180 attttttttt aaaactgttt gtttctcgttttaatttatt tttgcttgcc attccccact 240 tgaatcgggc cgacggcttg gggagattgctctacttccc caaatcactg tggattttgg 300 aaaccagcag aaagaggaaa gagagcaaga gc332 15 331 DNA Artificial Synthetic Construct 15 tcgcggaggc ttggggcagccgggtagctc ggagtcgtgg cgctgggggc tagcaccagc 60 gctctgtcgg gaggcgcagcggttaggtgg accgtcagcg gactcaccgg ccagggcgct 120 cggtgctgga atttgatattcattgatccg ggtttatccc tcttcttttt tcttaaacat 180 ttttttttaa aactgtattgtttctcgttt taattatttt tgcttgccat tccccacttg 240 aatcgggccg acggcttggggagattgctc tactccccaa atcactgtgg attttggaaa 300 ccagcagaaa gaggaaagaggtagcaagag c 331 16 330 DNA Artificial Synthetic Construct 16 tcgcggaggcttggggcagc cgggtagctc ggaggtcgtg gcgctggggg ctagcaccag 60 cgctctgtcgggaggcgcag cggttaggtg gaccggtcag cggactcacc ggccagggcg 120 ctcggtgctggaatttgata ttcattgatc cgggttttat ccctcttctt ttttcttaaa 180 catttttttttaaaactgta ttgtttctcg ttttaattta tttttgcttg ccattcccca 240 cttgaatcgggccgacggct tggggagatt gctctacttc cccaaatcac tgtggatttt 300 ggaaaccagcagaaagagga aagaggtagc 330 17 329 DNA Artificial Synthetic Construct 17tcgcggaggc ttggggcagc cgggtagctc ggaggtcgtg gcgctggggg ctagcaccag 60cgctctgtcg ggaggcgcag cggttaggtg gaccggtcag cggactcacc ggccagggcg 120ctcggtgctg gaattttcat tgatccgggt tttatccctc ttcttttttc ttaaacattt 180ttttttaact gtattgtttc tcgttttaat ttatttttgc ttgccattcc ccacttgaat 240cgggccgacg gcttggggag attgctctac ttccccaaat cactgtggat tttggaaacc 300agcagaaaga ggaaagaggt agcaagagc 329 18 328 DNA Artificial SyntheticConstruct 18 tcgcggaggc ttggggcagc cgggtagctc ggaggtcgtg gcgctgggggctagcaccag 60 cgctctgtcg ggaggcgcag cggttaggtg gaccggtcag cggactcaccggccagggcg 120 ctcggtgctg gaatttgata ttcattgatc cgggttttat ccctcttcttttttcttaaa 180 catttttttt taaaactgta ttgtttctcg ttttaattta tttttgcttgccattcccca 240 cttgaatcgg gccgacggct tggctctact tccccaaatc actgtggattttggaaacca 300 gcagaaagag gaaagaggta gcaagagc 328 19 326 DNA ArtificialSynthetic Construct 19 tcgcggaggc ttggggcagc ggaggtcgtg gcgctgggggctagcaccag cgctctgtcg 60 ggaggcgcag cggttaggtg gaccggtcag cggactcaccggccagggcg ctcggtgctg 120 gaatttgata ttcattgatc cgggttttat ccctcttcttttttcttaaa catttttttt 180 taaaactgta ttgtttctcg ttttaattta tttttgcttgccattcccca cttgaatcgg 240 gccgacggct tggggagatt gctctacttc cccaaatcactgtggatttt ggaaaccagc 300 agaaagagga aagaggtagc aagagc 326 20 326 DNAArtificial Synthetic Construct 20 tcgcggaggc ttggggcagc cgggtagctcggaggtcgtg gcgctggggg ctagcaccag 60 cgctctgtcg ggaggcgcag cggttaggtggaccggtcag cggactcacc ggccagggcg 120 ctcggtgctg gaatttgata ttcattgatccgggttttat ccctcttctt ttttcttaaa 180 catttttttt taaaactgta ttgtttctcgttttaattta tttttgcttg ccattcccca 240 cttgaatcgg gccgacggct tggggagattgctctacttc cccaaatcac tgtggatttt 300 ggaaaccagc agaaagagga aagagg 326 21316 DNA Artificial Synthetic Construct 21 tcgcggaggc ttggggcagccgggtagctc ggaggtcgtg gcgctggggg ctagcaccag 60 cgctctgtcg ggaggcgcagcggttaggtg gaccggtcag cggactcacc ggccagggcg 120 ctcggtgctg gaatttgatattcattgatc cgggttttat ccctcttctt ttttcttaaa 180 catttttttt taaaactgtattgtttctcg ttttaattta tttttgcttg ccattcccca 240 cttgaatcgg gccgacggcttggggagatt gctctacttc cccaaatcac tgtggatttt 300 ggaaaccagc agaaag 316 22306 DNA Artificial Synthetic Construct 22 ttggggcagc cgggtagctcggaggtcgtg gcgctggggg ctagcaccag cgctctgtcg 60 ggaggcgcag cggttaggtggaccggtcag cggactcacc ggccagggcg ctcggtgctg 120 gaatttgata ttcattgatccgggttttat ccctcttctt ttttcttaaa catttttttt 180 taaaactgta ttgtttctcgttttaattta tttttgcttg ccattcccca cttgaatcgg 240 gccgacggct tggggagattgctctacttc cccaaatcac tgtggatttt ggaaaccagc 300 agaaag 306 23 296 DNAArtificial Synthetic Construct 23 cgggtagctc ggaggtcgtg gcgctgggggctagcaccag cgctctgtcg ggaggcgcag 60 cggttaggtg gaccggtcag cggactcaccggccagggcg ctcggtgctg gaatttgata 120 ttcattgatc cgggttttat ccctcttcttttttcttaaa catttttttt taaaactgta 180 ttgtttctcg ttttaattta tttttgcttgccattcccca cttgaatcgg gccgacggct 240 tggggagatt gctctacttc cccaaatcactgtggatttt ggaaaccagc agaaag 296 24 286 DNA Artificial SyntheticConstruct 24 cgggtagctc ggaggtcgtg gcgctggggg ctagcaccag cgctctgtcgggaggcgcag 60 cggttaggtg gaccggtcag cggactcacc ggccagggcg ctcggtgctggaatttgata 120 ccctcttctt ttttcttaaa catttttttt taaaactgta ttgtttctcgttttaattta 180 tttttgcttg ccattcccca cttgaatcgg gccgacggct gctctacttccccaaatcac 240 tgtggatttt ggaaaccagc agaaagagga aagaggtagc aagagc 286 25276 DNA Artificial Synthetic Construct 25 gcgctggggg ctagcaccagcgctctgtcg ggaggcgcag cggttaggtg gaccggtcag 60 cggactcacc ggccagggcgctcggtgctg gaatttgata ttcattgatc cgggttttat 120 ccctcttctt ttttcttaaacatttttttt taaaactgta ttgtttctcg ttttaattta 180 tttttgcttg ccattccccacttgaatcgg gccgacggct tggggagatt gctctacttc 240 cccaaatcac tgtggattttggaaaccagc agaaag 276 26 266 DNA Artificial Synthetic Construct 26ggaggtcgtg gcgctggggg ctagcaccag cgctctgtcg ggaggcgcag cggttaggtg 60gaccggtcag cggactcacc ggccagggcg ctcggtgctg gaatttgata ccctcttctt 120ttttcttaaa catttttttt taaaactgta ttgtttctcg ccattcccca cttgaatcgg 180gccgacggct tggggagatt gctctacttc cccaaatcac tgtggatttt ggaaaccagc 240agaaagagga aagaggtagc aagagc 266

What is claimed:
 1. A nucleic acid construct comprising a nucleic acidsequence encoding a reporter polypeptide, wherein said nucleic acidsequence encoding a reporter polypeptide is operably linked to a NeRP,said NeRP is operably linked to a PTCRE, said PTCRE is not SEQ ID NO: 3,and expression of said reporter polypeptide is capable of beingmodulated relative to in an absence of said NeRP.
 2. The nucleic acidconstruct according to claim 1, wherein a NeRP1 (SEQ ID NO: 4) islocated within said nucleic acid sequence encoding a reporterpolypeptide.
 3. The nucleic acid construct according to claim 1, whereina NeRP1 (SEQ ID NO: 4) is located downstream of said nucleic acidsequence encoding a reporter polypeptide.
 4. The nucleic acid constructaccording to claim 1, wherein a NeRP1 (SEQ ID NO: 4) is located withinan intron and said intron is located within said nucleic acid sequenceencoding a reporter polypeptide.
 5. A nucleic acid molecule comprising anucleic acid sequence encoding a reporter polypeptide and a VEGF 5′ UTRnucleic acid sequence in an absence of SEQ ID NO:
 4. 6. The nucleic acidmolecule according to claim 5, wherein said VEGF 5′ UTR in an absence ofSEQ ID NO: 4 contains an internal ribosome entry site (“IRES”).
 7. Thenucleic acid molecule according to claim 5, further comprising ahigh-level mammalian expression vector capable of integrating randomlyinto the genome.
 8. The nucleic acid molecule according to claim 5,further comprising a high-level mammalian expression vector capable ofintegrating site-selectively into the genome.
 9. The nucleic acidmolecule according to claim 5, further comprising an episomal mammalianexpression vector.
 10. The nucleic acid molecule according to claim 5,wherein said nucleic acid sequence encoding a reporter polypeptidecontains an intron.
 11. The nucleic acid molecule according to claim 5,wherein said VEGF 5′ UTR in an absence of SEQ ID NO: 4 contains anintron.
 12. A nucleic acid molecule comprising a nucleic acid sequenceencoding a reporter polypeptide operably linked to a VEGF 5′ UTR in anabsence of SEQ ID NO:
 4. 13. The nucleic acid molecule according toclaim 12, wherein said nucleic acid sequence encoding a reporterpolypeptide contains an intron.
 14. A nucleic acid molecule comprising anucleic acid sequence encoding a reporter polypeptide, wherein saidnucleic acid sequence encoding a reporter polypeptide is operably linkeddownstream of a UTR containing a NeRP, and said UTR is not operablyupstream of SEQ ID NO:
 3. 15. A heterogeneous population of nucleic acidmolecules, wherein said heterogeneous population comprises a reporternucleic acid sequence, and said nucleic acid sequence encoding areporter polypeptide is operably linked to a VEGF 5′ UTR in an absenceof NeRP1 (SEQ ID NO: 4).
 16. The heterogeneous population of nucleicacid molecules according to claim 15, wherein said heterogeneouspopulation is isolated from a stable cell line.
 17. The heterogeneouspopulation of nucleic acid molecules according to claim 15, wherein saidpopulation is produced in vitro.
 18. The heterogeneous population ofnucleic acid molecules according to claim 15, wherein said population isused to produce polypeptides.
 19. The heterogeneous population ofnucleic acid molecules according to claim 15, wherein said heterogeneouspopulation is selected to exclude molecules with a 5′ cap.
 20. Theheterogeneous population of nucleic acid molecules according to claim15, wherein said heterogeneous population is poly-adenylated.
 21. Theheterogeneous population of nucleic acid molecules according to claim15, wherein said heterogeneous population is not poly-adenylated.
 22. Asubstantially purified nucleic acid molecule comprising between 95% and99% sequence identity with a nucleic acid molecule of SEQ ID NO: 3, afragment thereof, or a complement of either.
 23. A substantiallypurified nucleic acid molecule consisting of SEQ ID NO: 3, a fragmentthereof, or a complement of either.
 24. A substantially purified nucleicacid molecule consisting of a first nucleic acid sequence linked to aheterologous nucleic acid sequence encoding a polypeptide, wherein saidfirst nucleic acid sequence is selected from the group consisting of SEQID NO: 3, a fragment thereof, and a complement of either.
 25. Asubstantially purified nucleic acid molecule comprising between 95% and99% sequence identity with a nucleic acid molecule of SEQ ID NO: 4, afragment thereof, or a complement of either.
 26. A substantiallypurified nucleic acid molecule of a nucleic acid sequence selected froma group consisting of SEQ ID NO: 4, a fragment thereof, and a complementof either.
 27. A substantially purified nucleic acid molecule consistingof a first nucleic acid sequence linked to a heterologous nucleic acidsequence encoding a polypeptide, wherein said first nucleic acidsequence is selected from the group consisting of SEQ ID NO: 4, afragment thereof, and a complement of either.
 28. A method of making anucleic acid construct to screen for a compound comprising: a) providinga main ORF downstream of a promoter in said nucleic acid construct; b)operably linking a VEGF 5′ UTR in an absence of SEQ ID NO: 4 upstream ofsaid main ORF; and c) operably linking a VEGF 3′ UTR downstream of saidmain ORF.
 29. The method according to claim 28, wherein said main ORFencodes a reporter polypeptide.
 30. A method of screening in vivo for acompound that modulates UTR-dependent expression comprising: a)providing a cell having a nucleic acid molecule comprising a promoterupstream from a VEGF 5′ UTR in an absence of SEQ ID NO: 4, wherein saidVEGF 5′ UTR in an absence of SEQ ID NO: 4 is upstream from a nucleicacid sequence encoding a reporter polypeptide, and said nucleic acidsequence encoding a reporter polypeptide is upstream from a VEGF 3′ UTR;b) contacting said cell with a compound; c) producing a nucleic acidmolecule that contains a nucleic acid sequence encoding a reporterpolypeptide and does not contain SEQ ID NO: 4; and d) detecting saidreporter polypeptide.
 31. The method according to claim 30, wherein saidnucleic acid sequence encoding a reporter polypeptide is VEGF.
 32. Amethod of screening in vitro for a compound that modulates UTR-affectedexpression comprising: a) providing an in vitro translation system; b)contacting said in vitro translation system with a compound and anucleic acid molecule comprising a VEGF 5′ UTR in an absence of SEQ IDNO: 4, wherein said VEGF 5′ UTR in an absence of SEQ ID NO: 4 isupstream from a nucleic acid sequence encoding a reporter polypeptideand said nucleic acid sequence encoding a reporter polypeptide isupstream from a VEGF 3′ UTR; and c) detecting said reporter polypeptidein vitro.
 33. A method of expressing a nucleic acid molecule in a cellcomprising: a) providing a nucleic acid molecule to a cell, wherein saidnucleic acid molecule comprises a nucleic acid sequence encoding areporter polypeptide flanked by VEGF UTRs in an absence of SEQ ID NO: 4;and b) detecting said reporter polypeptide.
 34. The method according toclaim 33, wherein said nucleic acid molecule is produced by in vitrotranscription.
 35. The method according to claim 33, wherein saidnucleic acid molecule is a synthetically produced RNA molecule.
 36. Amethod of screening for a compound that modulates protein expressionthrough a main ORF-independent, UTR-affected mechanism comprising: a)growing a stable cell line having a reporter gene operably linked to aVEGF 5′ UTR in an absence of SEQ ID NO: 4; b) comparing said stable cellline in a presence of a compound relative to said stable cell line in anabsence of said compound; and c) selecting for said compound thatmodulates protein expression through a main ORF-independent,UTR-affected mechanism.
 37. A method of screening for a compound thatmodulates protein expression through a main ORF-independent,UTR-affected mechanism comprising: a) growing a stable cell line havinga main ORF operably linked to a VEGF 5′ UTR in an absence of SEQ ID NO:4; b) comparing said stable cell line in the presence of a compoundrelative to said stable cell line in the absence of said compound; andc) selecting for said compound that modulates protein expression througha main ORF-independent, UTR-affected mechanism.
 38. A method ofscreening for a compound that modulates protein expression through aVEGF-independent, UTR-affected mechanism comprising: a) substituting invivo a VEGF gene with a reporter gene, wherein a UTR consisting of SEQID NO: 3 is operably linked to said reporter gene, and said substitutionoccurs in a differentiated cell; b) growing said differentiated cell;and c) selecting for said compound that modulates protein expression ofsaid reporter gene through a main ORF-independent, UTR-affectedmechanism.
 39. A method of screening for a compound that modulatesprotein expression through a main ORF-independent, UTR-affectedmechanism comprising: a) substituting in vivo a main ORF with a reportergene, wherein a 5′ UTR is operably linked to said reporter gene andconsists of SEQ ID NO: 3, and said substitution occurs in adifferentiated cell; b) growing said differentiated cell; and c)selecting for said compound that modulates protein expression of saidreporter gene through a main ORF-independent, UTR-affected mechanism.40. A method of screening for a compound that modulates proteinexpression through a UTR-affected mechanism comprising: a) providing astable cell line having a reporter gene operably linked to a VEGF 5′ UTRin an absence of SEQ ID NO: 4, wherein said stable cell line mimicspost-transcriptional regulation of a VEGF gene found in vivo in presenceof said compound; b) maintaining said stable cell line; and c) selectingfor said compound that modulates protein expression of said reportergene through a UTR-affected mechanism.
 41. The method according to claim40, wherein said stable cell line is a transformed cell.
 42. A method ofscreening for a compound that modulates protein expression through aUTR-affected mechanism comprising: a) providing a stable cell linehaving a main ORF encoding a reporter polypeptide operably linked to aVEGF 5′ UTR in an absence of SEQ ID NO: 4, wherein said stable cell linemimics post-transcriptional regulation of a VEGF gene found in vivo in apresence of a compound; b) maintaining said stable cell line; and c)selecting for said compound that modulates protein expression of saidmain ORF through a UTR-affected mechanism.
 43. A method of screening fora compound that modulates protein expression through a UTR-affectedmechanism mediating the effect of a NeRP comprising: a) growing a stablecell line having a reporter gene operably linked to a 5′ VEGF UTR in anabsence of a NeRP1 (SEQ ID NO: 4); b) comparing said stable cell line ina presence of a compound relative to in an absence of said compound,wherein said compound does not modulate UTR-dependent expression if said5′ VEGF UTR in an absence of a NeRP1 (SEQ ID NO: 4) is operably linkedto a reporter gene; and c) selecting for said compound that modulatesprotein expression of said reporter gene through a UTR-affectedmechanism mediating the effect of a NeRP.
 44. The method according toclaim 43, wherein said c) selecting for said compound comprisescomparing said stable cell line in the presence of a compound relativeto in the absence of said compound, wherein said compound modulatesUTR-dependent expression if said reporter gene is operably linked tosaid 5′ VEGF UTR.
 45. A method of screening for a compound thatmodulates protein expression through a UTR-affected mechanism mediatingthe effect of a NeRP comprising: a) growing a stable cell line having amain ORF encoding a reporter polypeptide operably linked to a 5′ VEGFUTR in an absence of a NeRP1 (SEQ ID NO: 4); b) comparing said stablecell line in the presence of a compound relative to in the absence ofsaid compound, wherein said compound does not modulate UTR-dependentexpression if said 5′ VEGF UTR in an absence of a NeRP1 (SEQ ID NO: 4)is operably linked to a main ORF; and c) selecting for said compoundthat modulates protein expression of said main ORF through aUTR-affected mechanism mediating the effect of a NeRP.
 46. A method ofscreening for a compound that modulates protein expression through aUTR-affected mechanism mediating the effect of a NeRP comprising: a)growing a stable cell line having a reporter gene operably linked to aUTR having a NeRP1 (SEQ ID NO: 4); b) comparing said stable cell line ina presence of a compound relative to in an absence of said compound,wherein said compound modulates UTR-dependent expression if a NeRP1 (SEQID NO: 4) is operably linked to a reporter gene; and c) selecting forsaid compound that modulates protein expression of said reporter genethrough a UTR-affected mechanism mediating the effect of a NeRP.
 47. Amethod of screening for a compound that modulates protein expressionthrough a UTR-affected mechanism mediating the effect of a NeRPcomprising: a) growing a stable cell line having a main ORF encoding areporter polypeptide operably linked to a UTR having a NeRP1 (SEQ ID NO:4); b) comparing said stable cell line in a presence of a compoundrelative to in an absence of said compound, wherein said compoundmodulates UTR-dependent expression if a NeRP1 (SEQ ID NO: 4) is operablylinked to a main ORF encoding a reporter polypeptide; and c) selectingfor said compound that modulates protein expression of said main ORFencoding a reporter polypeptide through a UTR-affected mechanismmediating the effect of said NeRP.